Antibodies to alpha-synuclein and uses thereof

ABSTRACT

The invention provides antibodies that specifically bind human α-synuclein with a high affinity and reduces α-synuclein spreading in vivo, recombinant polypeptides comprising said antibodies or antigen-binding fragment thereof and methods for generating such polypeptides, as well as compositions and methods for generating α-synuclein antibodies, and methods of using α-synuclein antibodies for the treatment of diseases of the central nervous system, in particular alpha-synucleinopathies.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/170,468, filed Oct. 25, 2018(now U.S. Pat. No. 10,889,639), which isa continuation of U.S. application Ser. No. 15/611,416, filed Jun. 1,2017 (now U.S. Pat. No. 10,160,800), which claims the benefit ofpriority from U.S. Provisional Application No. 62/344,746, filed Jun. 2,2016 (now expired). The foregoing applications are incorporated hereinby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 24, 2018, isnamed 1848081-0002-091-103_SL.txt and is 45,626 bytes in size.

BACKGROUND

The present invention relates to α-synuclein antibodies and their use inthe prevention or treatment of disease, in particularalpha-synucleinopathies, and more particularly Parkinson's disease (PD).

Alpha-synucleinopathies, also known as Lewy body diseases (LBDs), are afamily of neurodegenerative diseases that all have at their corealpha-synuclein as the key pathological hallmark (Jellinger, Mov Disord(2003), 18 Suppl 6: S2-12; and Spillantini and Goedert, Ann N Y Acad Sci(2000), 920: 16-27; both of which are incorporated herein by reference).Alpha-synucleinopathies include Parkinson's disease (PD), dementia withLewy bodies (DLB) and multiple system atrophy (MSA).

PD is a slowly progressive age-related movement disorder affectinggreater than 1% of people over 65 years old. PD is the second mostcommon neurodegenerative condition after Alzheimer's disease.

A defining hallmark pathology of alpha-synucleinopathies are Lewy bodiesand Lewy neurites, which are insoluble inclusions of aggregated proteinsfound inside neurons of the brain revealed upon post-mortemhistopathological examination.

The presence of Lewy pathology and neuronal loss in non-motor brainregions such as the basal forebrain, mesopontine system, amygdala,neocortex, dorsal motor nucleus of the vagus nerve, olfactory bulbs,locus coeruleus, and the brainstem, may cause cognitive deficits anddementia, hyposmia, sleep disturbances including rapid eye movementsleep behaviour disorder (RBD), mood disorders including depression andanxiety, autonomic dysfunction including cardiovascular andgastrointestinal problems such as constipation, and fatigue andsomnolence. Some of these non-motor symptoms appear to characterise thepremotor or prodromal phase of the disease (Kalia et al. Lancet (2015),386(9996): 896-912; incorporated herein by reference).

The presence of Lewy pathology and neuronal loss in motor brain regions,including most notably the death of dopaminergic neurons in thesubstantia nigra, may cause resting tremor, rigidity, bradykinesia andpostural instability (Spillantini and Goedert, Ann N Y Acad Sci (2000),920: 16-27; incorporated herein by reference).

Alpha-synuclein (also called “α-synuclein” or “α-syn”) protein is themajor structural component of Lewy bodies and Lewy neurites.Alpha-synuclein is a small acidic protein made up of 140 amino acids (14kDa). Human natural wild-type alpha-synuclein has the amino acidsequence SEQ ID NO: 1 as described under UniProtKB accession numberP37840. Unless otherwise apparent from the context, reference toalpha-synuclein or its fragments includes the natural human wild-typeamino acid sequence indicated above, and human allelic variants thereof,in particular those associated with Lewy body disease (e.g., E46K, A30P,H50Q, G51D and A53T, where the first letter indicates the amino acid inSEQ ID NO: 1, the number is the codon position in SEQ ID NO: 1, and thesecond letter is the amino acid in the allelic variant). Such variantscan optionally be present individually or in any combination. Theinduced mutations E83Q, A90V, A76T, which enhance alpha-synucleinaggregation, can also be present individually or in combination witheach other and/or with human allelic variants E46K, A30P, H50Q, G51D andA53T. At the structural level, alpha-synuclein contains three distinctregions: an amphipathic N-terminal alpha-helix domain that has lipid andmembrane binding properties (residues 1-60), a central hydrophobicamyloid-binding domain that encodes the non-amyloid-beta component (NAC)of plaques (residues 61-95), and an acidic proline-rich C-terminal tail(residues 96-140). Residues 71-82 of the NAC domain are believed to bekey to the aggregation/fibrillation properties of alpha-synuclein byenabling the protein to switch from a random coil structure to abeta-sheet structure (Bisaglia et al. FASEB J (2009), 23(2): 329-40;incorporated herein by reference). Although the C-terminal domain isfree of significant secondary structure it contains a keyphosphorylation site at residue Ser129 and a number of tyrosine residuesthat are nitrated in cytosolic alpha-synuclein inclusions. N-terminaland C-terminal truncated forms of alpha-synuclein also exist.Post-translational modifications to the protein can affectalpha-synuclein aggregation and toxicity (Oueslati et al. Prog Brain Res(2010), 183: 115-45; incorporated herein by reference).

Alpha-synuclein is abundant in the central nervous system (CNS)/brainwhere it is found both intracellularly in neurons and glia and alsoextracellularly in cerebrospinal fluid (CSF) (Mollenhauer et al. JNeural Transm (2012), 119(7): 739-46; incorporated herein by reference)and the interstitial fluid (ISF) that bathes and surrounds the cells ofthe brain (Emmanouilidou et al. PLoS One (2011), 6(7): e22225;incorporated herein by reference). Alpha-synuclein is a synaptic proteinpredominantly expressed in neurons of the neocortex, hippocampus,substantia nigra, thalamus, and cerebellum (Iwai et al. Neuron (1995),14: 467-475; incorporated herein by reference). Under physiologicalconditions, it is located in neuronal synaptic terminals and isspecifically up-regulated at presynaptic terminals duringacquisition-related synaptic rearrangement (Fortin et al. J Neurosci(2005), 25: 10913-10921; incorporated herein by reference).

In-vitro studies have shown that alpha-synuclein monomers may form thestarting point for the aggregation process. The monomer can aggregateinto a variety of small oligomeric species that are then stabilised bybeta-sheet interactions, going on to form protofibrils which canpolymerise into insoluble fibrillary structures reminiscent of thoseidentified in Lewy bodies (Cremades et al. Cell (2012), 149(5): 1048-59;incorporated herein by reference).

Under pathological conditions, aberrant alpha-synuclein aggregation maybe key to the pathological changes seen in alpha-synucleinopathies(Lashuel et al. Nature (2002), 418: 291; and Tsigelny et al. FEBSJournal (2007), 274: 1862-1877; both of which are incorporated herein byreference). In vitro and in vivo studies have shown that the neurotoxiceffects of alpha-synuclein appear to be elicited by small solubleoligomeric conformers or protofibrils (Winner et al. Proc Natl Acad SciU S A (2011), 108(10): 4194-9; and Danzer et al. J Neurosci (2007),27(34): 9220-32; both of which are incorporated herein by reference).While fibrillar aggregates of alpha-synuclein are characteristic of PD,oligomeric forms of alpha-synuclein are the toxic species (Danzer et al.J Neurosci (2007), 27(34): 9220-32; Lashuel et al. Nature (2002), 418:291; and Winner et al. Proc Natl Acad Sci USA (2011), 108: 4194-4199;each of which are incorporated herein by reference).

Alpha-synuclein oligomers can be released to the extracellularenvironment and taken up by neighboring cells in a “propagation”mechanism (Angot and Brundin, Parkinsonism Relat Disord (2009), 15 Suppl3: S143-147; Desplats et al. Proc Natl Acad Sci USA (2009), 106:13010-13015; and Lee et al. J Biol Chem (2010), 285: 9262-9272; each ofwhich are incorporated herein by reference). Aggregates ofalpha-synuclein can propagate misfolding through a prion-like spreadingmechanism (Lee et al. Nat Rev Neurol (2010), 6: 702-706; Luk et al. JExp Med (2012), 209(5): 975-86; and Luk et al. Science (2012),338(6109): 949-53; each of which are incorporated herein by reference).Alpha-synuclein can therefore induce neurodegeneration by eitheroligomer toxicity or propagation and prion-like spreading.

It is now well established and accepted that cells including neurons cansecrete various forms of alpha-synuclein (monomers, oligomers,aggregates) under normal conditions and also under conditions ofcellular stress, the secretion of monomeric and aggregated forms ofalpha-synuclein is elevated under conditions of cellular stress, andthrough this release of alpha-synuclein into the extracellular milieu,pathological transmissible forms of alpha-synuclein may be propagatedbetween neurons (Recasens and Dehay, Front Neuroanat (2014), 8: 159;incorporated herein by reference).

The effects of alpha-synuclein in PD may extend beyond the immediatedamage to vulnerable neuronal cells. Like most neurodegenerativediseases there is also a pro-inflammatory cellular response observed(Lee et al. J Biol Chem (2010), 285: 9262-9272; incorporated herein byreference). Circulating alpha-synuclein and/or activated astrocytes canactivate microglia, leading to increased generation of reactive oxygenspecies, nitric oxide and cytokine production, and further exacerbatingneurodegeneration (Lee et al. J Biol Chem (2010), 285: 9262-9272;incorporated herein by reference).

A variety of different experimental models have demonstratedcell-to-cell transmission of alpha-synuclein in cultured cells, or invivo spreading and propagation of alpha-synuclein pathologies. Lewy bodypathology has been observed within embryonic mesencephalic neuronalgrafts more than 10 years after the grafts were therapeuticallytransplanted into the striatum of PD patients. Specifically, graftedneurons contained a number of Lewy body-like inclusions that stainedpositively for alpha-synuclein, indicating that host-to-grafttransmission of alpha-synuclein pathology had occurred (Li et al. NatMed (2008), 14(5): 501-3; and Kordower et al. Nat Med (2008), 14(5):504-6; both of which are incorporated herein by reference).

Further, preformed recombinant alpha-synuclein fibrils andalpha-synuclein oligomers can be internalised by cultured cells andneurons, and the direct transfer of alpha-synuclein from donor torecipient cells with the formation of alpha-synuclein inclusions similarto Lewy pathology has been demonstrated (Danzer et al. J Neurosci(2007), 27(34): 9220-32; Volpicelli-Daley et al. Neuron (2011), 72(1):57-71; and Luk et al. Proc Natl Acad Sci U S A (2009), 106(47): 20051-6;each of which are incorporated herein by reference). Injection ofpreformed synthetic alpha-synuclein fibrils or Lewy body-likealpha-synuclein containing material extracted from the brains of agedalpha-synuclein transgenic mice into the brains of asymptomaticrecipient mice promotes the formation of Lewy body-like pathology inhost neurons of the recipient animals along with neurodegeneration andneurological deficits (Luk et al. J Exp Med (2012), 209(5): 975-86; andLuk et al. Science (2012), 338(6109): 949-53; both of which areincorporated herein by reference). Alpha-synuclein containing Lewy bodyextracts isolated from PD brains inoculated into the substantia nigra orstriatum of macaque monkeys and mice is rapidly taken up by host cells(within 24 hours) followed by a slower loss of striatal dopaminergicterminals, with cell loss evident after more than a year (Recasens etal. Ann Neurol (2014), 75(3): 351-62; incorporated herein by reference).Similarly, inoculation of mice with brain homogenates derived frompatients with the synucleinopathies DLB or MSA triggers alpha-synucleinLewy-like pathology in the host mice (Watts et al. Proc Natl Acad Sci US A (2013), 110(48): 19555-60; and Masuda-Suzukake et al. Brain (2013),136(Pt 4): 1128-38; both of which are incorporated herein by reference).Finally, transfer and transmission of both monomeric and oligomericalpha-synuclein from the olfactory bulb to interconnected brainstructures has been demonstrated in mice (Rey et al. Acta Neuropathol(2013), 126(4): 555-73; incorporated herein by reference).

Passive immunotherapy approaches with antibodies targetingalpha-synuclein have been tested in numerous preclinicalalpha-synucleinopathy mouse models (Lawand et al. Expert Opin TherTargets (2015): 1-10; incorporated herein by reference). Specifically, astudy using a monoclonal antibody directed against alpha-synuclein (9E4)has shown in vivo clearance of alpha-synuclein aggregates and pathology,behavioural motor improvements, and neuroprotective effects (WO2014/058924; which is incorporated herein by reference).

Further studies using passive immunisation of alpha-synuclein transgenicmice developed as experimental models of PD/DLB, with the 9E4 monoclonalantibody have shown the antibody to clear alpha-synuclein pathology,decrease synaptic and axonal deficits, abrogate loss of striataltyrosine hydroxylase fibres, and significantly reduce memory deficitsand motor function impairments (Games et al. J Neurosci (2014), 34(28):9441-54; Bae et al. J Neurosci (2012), 32(39): 13454-69; and Masliah etal. PLoS One (2011), 6(4): e19338; each of which are incorporated hereinby reference). Further it has been demonstrated that passiveadministration of anti-alpha-synuclein monoclonal antibodies inwild-type mice that were injected intrastriatally with syntheticalpha-synuclein preformed fibrils (pffs) led to robust reduction in Lewypathology, prevention of dopamine neuron loss in the substantia nigra,and a significant improvement in motor impairments that are manifest inthe mouse model after pffs treatment (Tran et al. Cell Rep (2014), 7(6):2054-65; incorporated herein by reference).

Additionally, one of the major challenges associated with treatingdisorders of the CNS with large molecule therapeutics, such asantibodies, is getting these drugs into the affected tissue. The passageof large molecules into the brain and spinal cord is largely restrictedby the blood-brain barrier (BBB). The BBB protects and regulates thehomeostasis of the brain and prevents the free passage of molecules intomost parts of the brain, thereby limiting the treatment of many braindiseases. Transport of essential molecules such as nutrients, growthfactors, and hormones is achieved via a series of specific transportersand receptors that regulate passage across the brain endothelial cells.The delivery of biologics and other drugs to the brain thereforerepresents a significant challenge. Additionally, transport mechanismsappear to exist that rapidly remove antibodies from the brain,presumably to prevent inflammatory responses due to engagement of Fcwith effector ligands that promote a pro-inflammatory response.

Over the last decade, reports of antibody transport across the BBB haveemerged where binding to the extracellular domain of the transportermolecules facilitates transcytosis of the receptor antibody complexacross the endothelial cell layer.

The BBB is mainly comprised of brain capillary endothelial cells, whichhave specialized characteristics, such as tight junctions, to limittransport of molecules into the brain (Reese et al. 1967, J. Cell Biol.34: 207-217; Brightman et al. 1969, J. Cell Biol. 40: 648-677; Rubin etal. 1999, Ann. Rev. Neurosci. 22: 11-28), although other cell types,such as pericytes, astrocytes, and neuronal cells, also play animportant role in the function of the BBB. Typically, less than 0.1% ofa peripherally dosed antibody reaches the brain (Boado et al. 2010, Mol.Pharm. 7: 237-244; Pepinsky et al. 2011, Nat. Neurosci. 8: 745-751). TheBBB functions as a physical, metabolic and immunological barrier(Gaillard et al. 2003, Microvasc. Res. 65: 24-31).

Antibody transport across the BBB can be enhanced by triggering receptormediated transcytosis on brain endothelial cells. Through this process,engagement of antigens on the luminal side of the endothelial cell caninduce the internalization and shuttling of the antibody across thecell, and then its subsequent release into the tissue.

Current drug therapies for PD are mainly focused on treating themotor-related symptoms of the disease. There are currently no marketedor available therapies that can treat or prevent alpha-synucleinopathy.

Accordingly, there is a need in the art for a therapy for treatingalpha-synucleinopathies, particularly in humans.

SUMMARY OF THE INVENTION

The present invention is concerned with isolated antibodies to humanalpha-synuclein. The invention provides antibodies or antigen-bindingfragments thereof that have one or more of the functional properties ofthe aslo0452 ngl-3 antibody. For example:

The invention provides an antibody or antigen-binding fragment thereofthat specifically binds the C-terminal region of human α-synuclein. Theinvention provides an antibody or antigen-binding fragment thereof thatspecifically binds the region comprising about amino acid 102 to aboutamino acid 130 of human α-synuclein (e.g., SEQ ID NO: 1). In someembodiments, the antibody or antigen-binding fragment thereofspecifically binds to a region comprising about amino acid 120 to aboutamino acid 130 of human α-synuclein (SEQ ID NO: 1). In some embodiments,the antibody or antigen-binding fragment thereof binds to an epitopethat is not the same as the antibody bound by the 9E4 antibody.

The invention provides an antibody or antigen-binding fragment thereofthat binds human α-synuclein but not human β-synuclein or humanγ-synuclein.

The invention provides an antibody or antigen-binding fragment thereofthat binds to human, rat and cynomolgus α-synuclein.

The invention provides an antibody or antigen-binding fragment thereofthat binds to human α-synuclein with high affinity. In one embodiment,the antibody, or antigen-binding fragment thereof, of the inventionbinds to alpha-synuclein with a K_(D) of less than 500 picoMolar (pM),less than 400 pM, less than 300 pM, less than 200 pM, less than 150 pM,less than 120 pM, less than 110 pM or 106 pM or less as measured forexample using Octet analysis. In one embodiment, the antibody, orantigen-binding fragment thereof, of the invention binds toalpha-synuclein with a K_(D) of less than 400 picoMolar (pM), less than300 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than120 pM, less than 110 pM, less than 100 pM, less than 80 pM or 74 pM orless as measured for example using KinExA analysis.

The invention provides an antibody or antigen-binding fragment thereofthat binds to native endogenous human α-synuclein.

The invention provides an antibody or antigen-binding fragment thereofthat binds to monomeric forms of human α-synuclein.

The invention provides an antibody or antigen-binding fragment thereofthat binds to aggregates of human α-synuclein.

The invention provides an antibody or antigen-binding fragment thereofthat binds disease relevant, pathological forms of α-synuclein.

The invention provides an antibody or antigen-binding fragment thereofof the invention that reduces α-synuclein levels in the braininterstitial fluid. In particular, the antibody or antigen-bindingfragment thereof according to the invention reduces free unboundα-synuclein levels in the brain interstitial fluid.

The invention provides an antibody or antigen-binding fragment thereofof the invention that reduces α-synuclein levels in the cerebrospinalfluid. In particular, the antibody or antigen-binding fragment thereofaccording to the invention reduces free unbound α-synuclein levels inthe cerebrospinal fluid.

As used herein, the terms “free unbound α-synuclein” refer toα-synuclein that is not bound to the antibody or antigen-bindingfragment thereof according to the invention. Said free unboundα-synuclein can apply to α-synuclein in its monomeric or oligomericform, or in aggregated form. These terms generally apply to anypathologic form of α-synuclein.

The invention provides an antibody or antigen-binding fragment thereofthat reduces α-synuclein spreading in vivo.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention competes with antibody aslo0452 ngl-3 for bindingto human α-synuclein.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention binds to the same epitope on human α-synuclein asantibody aslo0452 ngl-3.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention derives from antibody asyn0087 comprising avariable heavy chain region (VH) of amino acid sequence of SEQ ID NO: 2and a variable light chain region (VL) of amino acid sequence SEQ ID NO:3, as disclosed herewith.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the present invention derives from antibody asyn0087, whereinsaid antibody or antigen-binding fragment has a K_(D) of less than 500mM and binds the same epitope as any one of antibodies asyn0087,aslo0452 ngl-3 and aslo0543, described herewith.

As used herewith, “H-CDR” stands for a complementary determining region(CDR) on the heavy chain region, and “L-CDR” stands for a complementarydetermining region (CDR) on the light chain region of an antibody orantigen-binding fragment thereof.

In one embodiment, the antibody or antigen-binding fragment thereofaccording to the invention comprises at least one CDR selected from:

-   -   6 H-CDR1 of SEQ ID NO: 5,    -   (ii) H-CDR2 of SEQ ID NO: 6,    -   (iii) H-CDR3 of SEQ ID NO: 7,    -   (iv) L-CDR1 of SEQ ID NO: 9,    -   (v) L-CDR2 of SEQ ID NO: 10,    -   (vi) L-CDR3 of SEQ ID NO: 11.

In a further embodiment, the CDR3 of the heavy chain of the antibody orantigen-binding fragment thereof according to the invention is CDR3 ofSEQ ID NO: 16 of the heavy chain of antibody aslo0452 ngl-3; and/or theCDR3 of the light chain of the antibody or antigen-binding fragmentthereof according to the invention is CDR3 of SEQ ID NO: 21 of the lightchain of antibody aslo0452 ngl-3.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention has at least one, at least two, at least three, atleast four, at least five or all of the CDRs selected from the CDRs ofantibody aslo0452 ngl-3, i.e. at least one CDR selected from any one ofSEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO:10, SEQ ID NO: 21.

In one embodiment, the CDR3 of the heavy chain of the antibody orantigen-binding fragment thereof of the present invention is CDR3 of theheavy chain of antibody aslo0452 ngl-3; and/or the CDR3 of the lightchain of the antibody or antigen-binding fragment thereof of the presentinvention is CDR3 of the light chain of antibody aslo0452 ngl-3.

In one embodiment, the CDR3 of the heavy chain of the antibody orantigen-binding fragment thereof of the present invention is CDR3 of theheavy chain of antibody aslo0452 ngl-3.

In one embodiment, the CDR3 of the light chain of the antibody orantigen-binding fragment thereof of the present invention is CDR3 of thelight chain of antibody aslo0452 ngl-3.

In one embodiment, the CDR3 of the heavy chain of the antibody orantigen-binding fragment thereof of the present invention is CDR3 of theheavy chain of antibody aslo0452 ngl-3 and the CDR3 of the light chainof the antibody or antigen-binding fragment thereof of the presentinvention is CDR3 of the light chain of antibody aslo0452 ngl-3.

The present invention provides an antibody or antigen-binding fragmentthereof having the six CDRs of antibody aslo0452 ngl-3.

Thus, in one embodiment, the antibody, or antigen-binding fragmentthereof according to the invention comprises:

-   -   a) three heavy chain CDRs having sequences:        -   (i) H-CDR1 of SEQ ID NO: 5,        -   (ii) H-CDR2 of SEQ ID NO: 15; and        -   (iii) H-CDR3 of SEQ ID NO: 16, and    -   b) three light chain CDRs having sequences:        -   (i) L-CDR1 of SEQ ID NO: 20,        -   (ii) L-CDR2 of SEQ ID NO: 10, and        -   (iii) L-CDR3 of SEQ ID NO: 21.

The present invention provides an antibody or antigen-binding fragmentthereof of the present invention comprising a variable heavy chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the nucleotide sequence defined by SEQ ID NO: 13 and a variable lightchain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the nucleotide sequence defined by SEQ ID NO: 18.

The present invention provides an antibody or antigen-binding fragmentthereof of the present invention comprising a variable heavy chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the amino acid sequence defined by SEQ ID NO: 14 and a variable lightchain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the amino acid sequence defined by SEQ ID NO: 19.

The present invention provides an antibody or antigen-binding fragmentthereof of the present invention comprising a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 14 and a variable lightchain having the amino acid sequence of SEQ ID NO: 19.

In a particular embodiment, the antibody or antigen-binding fragmentthereof according to the invention comprises a variable heavy chainhaving a sequence defined by SEQ ID NO: 4 and a variable light chainhaving a sequence defined by SEQ ID NO: 8.

In a further particular embodiment, the antibody or antigen-bindingfragment thereof according to the invention comprises a variable heavychain having a sequence defined by SEQ ID NO: 4 and a variable lightchain having a sequence defined by SEQ ID NO: 8, and binds to humanα-synuclein with a K_(D) of less than 500 pM and binds the same epitopeas asyn0087, aslo0452 ngl-3 or aslo0543.

In another embodiment, the antibody or antigen-binding fragment thereofaccording to the invention comprises a variable heavy chain having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to thesequence defined by SEQ ID NO: 14 and a variable light chain having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to thesequence defined by SEQ ID NO: 19.

In a particular embodiment, the antibody or antigen-binding fragmentthereof according to the invention comprises a variable heavy chainhaving a sequence defined by SEQ ID NO: 14 and a variable light chainhaving a sequence defined by SEQ ID NO: 19.

In a further embodiment, the antibody, or antigen-binding fragmentthereof according to the invention comprises a variable heavy chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the sequence defined by SEQ ID NO: 14 and a variable light chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the sequence defined by SEQ ID NO: 19 and further comprises:

-   -   a) three heavy chain CDRs having sequences:        -   (i) H-CDR1 of SEQ ID NO: 5,        -   (ii) H-CDR2 of SEQ ID NO: 15; and        -   (iii) H-CDR3 of SEQ ID NO: 16, and    -   b) three light chain CDRs having sequences:        -   (i) L-CDR1 of SEQ ID NO: 20,        -   (ii) L-CDR2 of SEQ ID NO: 10; and        -   (iii) L-CDR3 of SEQ ID NO: 21.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a variable heavy chain having a nucleotidesequence defined by SEQ ID NO: 13 and a variable light chain having anucleotide sequence defined by SEQ ID NO: 18.

The present invention provides an antibody or antigen-binding fragmentthereof comprising a variable heavy chain having an amino acid sequencedefined by SEQ ID NO: 14 and a variable light chain having an amino acidsequence defined by SEQ ID NO: 19.

Also provided is an antibody or antigen-binding fragment thereof of thepresent invention comprising a heavy chain having an amino acid sequencedefined by SEQ ID NO: 12 and a light chain having an amino acid sequencedefined by SEQ ID NO: 17.

In another embodiment, the antibody or antigen-binding fragment thereofaccording to the invention comprises a variable heavy chain having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to thesequence defined by SEQ ID NO: 24 and a variable light chain having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to thesequence defined by SEQ ID NO: 30.

In a particular embodiment, the antibody or antigen-binding fragmentthereof according to the invention comprises a variable heavy chainhaving a sequence defined by SEQ ID NO: 24 and a variable light chainhaving a sequence defined by SEQ ID NO: 30.

In a further embodiment, the antibody, or antigen-binding fragmentthereof according to the invention comprises a variable heavy chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the sequence defined by SEQ ID NO: 24 and a variable light chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the sequence defined by SEQ ID NO: 30 and further comprises:

-   -   c) three heavy chain CDRs having sequences:        -   (iv) H-CDR1 of SEQ ID NO: 25,        -   (v) H-CDR2 of SEQ ID NO: 26; and        -   (vi) H-CDR3 of SEQ ID NO: 27, and    -   d) three light chain CDRs having sequences:        -   (iv) L-CDR1 of SEQ ID NO: 31,        -   (v) L-CDR2 of SEQ ID NO: 32; and        -   (vi) L-CDR3 of SEQ ID NO: 33.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a variable heavy chain having a nucleotidesequence defined by SEQ ID NO: 24 and a variable light chain having anucleotide sequence defined by SEQ ID NO: 30.

The present invention provides an antibody or antigen-binding fragmentthereof comprising a variable heavy chain having an amino acid sequencedefined by SEQ ID NO: 24 and a variable light chain having an amino acidsequence defined by SEQ ID NO: 30.

Also provided is an antibody or antigen-binding fragment thereof of thepresent invention comprising a heavy chain having an amino acid sequencedefined by SEQ ID NO: 22 and a light chain having an amino acid sequencedefined by SEQ ID NO: 28.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention as defined anywhere above is an IgA, IgD, IgE,IgM, IgG such as IgG1, IgG2, IgG3, or IgG4 antibody or antigen-bindingfragment thereof.

In another embodiment, the antibody or antigen-binding fragment thereofof the present invention has a modified Fc region. Suitablemodifications are well known to those skilled in the art and may includeinter alia modifications to increase or decrease half-life, ablate,reduce or enhance effector function, provide substituted cysteines withfree thiols for conjugation. Examples of such modifications are YTE toincrease half-life and/or TM to reduce effector function. In someembodiments, any of the antibodies or antigen-binding fragmentsdisclosed herein comprise mutations M252Y/S254T/T256E (YTE) in the Fcregion of the antibody (Dall' Acqua et al., 2006, J. Biol. Chem,281:23514-23524). In some embodiments, any of the antibodies orantigen-binding fragments disclosed herein comprise a triple mutation(abbreviated herein as “TM”) in the Fc region corresponding to theL234F/L235E/P331S mutation disclosed in Oganesyan et al. ActaCrystallogr D Biol Crystallogr, (2008) 64: 700-704. In an embodiment theantibody or antigen-binding fragment thereof of the present inventionmay be an IgG1 TM antibody or antigen-binding fragment thereof. Inanother embodiment the antibody or antigen-binding fragment thereof ofthe present invention may comprise a Fc region having YTE mutations.

In another embodiment the antibody, or antigen-binding fragment thereof,of the present invention may be coupled to a blood-brain barrier (BBB)transporter moiety, wherein the BBB transporter moiety is capable oftransporting the antibody, or antigen binding fragment thereof, acrossthe BBB.

In an embodiment the BBB transporter moiety may be an antibody. In anembodiment the BBB antibody may form a multispecific construct with ananti-α-syncuclein antibody or antigen binding fragment thereof. The BBBtransporter moiety may comprise an immunoglobulin variable heavy chaincomplementarity-determining region-1 (VH-CDR1), an immunoglobulinvariable heavy chain complementarity-determining region-2 (VH-CDR2), animmunoglobulin variable heavy chain complementarity-determining region-3(VH-CDR3), an immunoglobulin variable light chaincomplementarity-determining region-1 (VL-CDR1), an immunoglobulinvariable light chain complementarity-determining region-2 (VL-CDR2), andan immunoglobulin variable light chain complementarity-determiningregion-3 (VL-CDR3); wherein the VH-CDR1 comprises SEQ ID NO. 40 or 49,VH-CDR2 comprises SEQ ID NO. 41 or 50, VH-CDR3 comprises SEQ ID NO. 42or 51, VL-CDR1 comprises SEQ ID NO. 36, 44 or 53, VL-CDR2 comprises SEQID NO. 37, 45 or 54 and VL-CDR3 comprises SEQ ID NO. 38, 46 or 55.

In some embodiments, the transporter moiety comprises an immunoglobulinvariable heavy chain (VH) region comprising SEQ ID NO: 47 or SEQ ID NO:39. In some embodiments, the transporter moiety comprises animmunoglobulin variable light chain (VL) region comprising SEQ ID NO:43.

Additionally, the transporter moiety can be selected from a completeantibody, an Fv fragment, a Fab fragment, a Fab' fragment, a F(ab')2fragment, a disulfide-linked (dsFv) fragment, a single chain Fv (scFV)fragment, an sc(Fv)2 fragment, a diabody, a triabody, a tetrabody, aminibody, and a single chain antibody. In a particular embodiment, thetransporter moiety comprises an scFV fragment comprising a VH domain anda VL domain fused together via a linker. In some instances, the linkercan be (Gly4Ser)^(n) (SEQ ID NO: 56), wherein n is a positive integerselected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

In some embodiments, any transporter molecule of the invention can becombined as described herein with any alpha-synuclein binding moleculeof the invention to provide a multi-specific binding molecule of theinvention.

The invention provides the antibody or antigen-binding fragment thereofof the present invention as defined anywhere above, for use as amedicament.

The invention also provides the antibody or antigen-binding fragmentthereof of the present invention as defined anywhere above for use inthe prevention or treatment of an α-synucleinopathy.

In one embodiment, the α-synucleinopathy is selected from Parkinson'sdisease (PD), dementia with Lewy bodies (DLB), and multiple systematrophy (MSA).

In one embodiment, the α-synucleinopathy is Parkinson's disease (PD).

The invention provides a method of treating or preventing a disease, inparticular a disease associated with the central nervous system, in apatient, the method comprising administering to the patient the antibodyor antigen-binding fragment thereof of the present invention as definedanywhere above.

In one embodiment, the disease is an α-synucleinopathy.

In one embodiment, the α-synucleinopathy is selected from Parkinson'sdisease (PD), dementia with Lewy bodies (DLB), and multiple systematrophy (MSA).

In one embodiment, the α-synucleinopathy is Parkinson's disease (PD).

The invention provides a pharmaceutical composition comprising theantibody or antigen-binding fragment thereof of the present invention asdefined anywhere above, and a pharmaceutically acceptable excipient.

The phrase “pharmaceutically acceptable excipient” includes any and allsolvents, dispersion media, coatings, antibacterial agents andantifungal agents, isotonic agents, and absorption delaying agents, andthe like, that are compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions. The pharmaceutical compositions may also be included in acontainer, pack, or dispenser together with instructions foradministration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. The administration may, for example, be intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous ortransdermal.

The invention provides an isolated nucleic acid molecule encoding theantibody or antigen-binding fragment thereof of the present invention asdefined anywhere above.

In a particular embodiment, the present invention provides an isolatednucleic acid molecule comprising SEQ ID NO: 13 and/or SEQ ID NO: 18.

In another particular embodiment, the present invention provides anisolated nucleic acid molecule comprising SEQ ID NO: 23 and/or SEQ IDNO: 29.

Once provided with this information, one of skill in the art couldreadily obtain nucleic acid molecules encoding the disclosed antibodiesor antigen-binding fragments thereof. The nucleic acids may comprise DNAor RNA and may be wholly or partially synthetic or recombinant.Reference to a nucleotide sequence encompasses a DNA molecule with thespecified sequence, and encompasses a RNA molecule with the specifiedsequence in which U is substituted for T, unless context requiresotherwise.

The nucleic acids molecules of the invention comprise a coding sequencefor a CDR, a VH domain, and/or a VL domain disclosed herein.

The present disclosure also provides constructs in the form of plasmids,vectors, phagemids, transcription or expression cassettes which compriseat least one nucleic acid molecule encoding the antibody orantigen-binding fragment thereof of the present invention as definedanywhere above, in particular encoding a CDR, a VH domain, and/or a VLdomain disclosed herein.

The disclosure further provides a host cell which comprises one or moreconstructs as above.

Also provided are nucleic acids encoding any one or more CDR (H-CDR1,H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3), VH or VL domain disclosedherein, as well as methods of making of the encoded products. The methodcomprises expressing the encoded product from the encoding nucleic acid.Expression may be achieved by culturing under appropriate conditionsrecombinant host cells containing the nucleic acid. Following productionby expression a VH or VL domain, or specific binding member may beisolated and/or purified using any suitable technique, then used asappropriate.

Antigen-binding fragments, VH and/or VL domains and encoding nucleicacid molecules and vectors may be isolated and/or purified from theirnatural environment, in substantially pure or homogeneous form, or, inthe case of nucleic acid, free or substantially free of nucleic acid orgenes of origin other than the sequence encoding a polypeptide with therequired function.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known in the art. For cells suitable forproducing antibodies, see Gene Expression Systems, Academic Press, eds.Fernandez et al., 1999. Briefly, suitable host cells include bacteria,plant cells, mammalian cells, and yeast and baculovirus systems.Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, NS0 mouse myeloma cells, and manyothers. A common bacterial host is E. coli. Any protein expressionsystem compatible with the invention may be used to produce thedisclosed antibodies. Suitable expression systems include transgenicanimals described in Gene Expression Systems, Academic Press, eds.Fernandez et al., 1999.

Suitable vectors can be chosen or constructed, so that they containappropriate regulatory sequences, including promoter sequences,terminator sequences, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Vectors may be plasmidsor viral, e.g., phage, or phagemid, as appropriate. For further detailssee, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. Many knowntechniques and protocols for manipulation of nucleic acid, for example,in preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, 2nd Edition, eds. Ausubel et al., John Wiley & Sons, 1992.

A further aspect of the disclosure provides a host cell comprising anucleic acid as disclosed here, in particular a vector comprising anucleic acid molecule encoding the antibody or antigen-binding fragmentthereof of the present invention as defined anywhere above.

A still further aspect provides a method comprising introducing suchnucleic acid into a host cell. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.,vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage. The introductionof the nucleic acid into the cells may be followed by causing orallowing expression from the nucleic acid, e.g., by culturing host cellsunder conditions for expression of the gene.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING

The present invention will now be described in more detail withreference to the attached Figures and sequence listing, in which areshown:

Key to SEQ ID NOs:

TABLE 1 Affinity determination of key anti-α-synuclein antibodies forhuman α-syn performed on two affinity measurement platforms. SEQ NameDescription ID NO: human alpha synuclein amino acid sequence 1 asyn0087VH amino acid sequence 2 VL amino acid sequence 3 General sequence VHamino acid sequence 4 of an antibody H-CDR1 amino acid sequence 5derived from asyn0087 H-CDR2 amino acid sequence 6 H-CDR3 amino acidsequence 7 VL amino acid sequence 8 L-CDR1 amino acid sequence 9 L-CDR2amino acid sequence 10 L-CDR3 amino acid sequence 11 aslo0452 ngl-3Heavy chain amino acid sequence 12 VH nucleotide sequence 13 VH aminoacid sequence 14 H-CDR1 amino acid sequence 5 H-CDR2 amino acid sequence15 H-CDR3 amino acid sequence 16 Light chain amino acid sequence 17 VLnucleotide sequence 18 VL amino acid sequence 19 L-CDR1 amino acidsequence 20 L-CDR2 amino acid sequence 10 L-CDR3 amino acid sequence 21aslo0543 Heavy chain amino acid sequence 22 VH nucleotide sequence 23 VHamino acid sequence 24 H-CDR1 amino acid sequence 25 H-CDR2 amino acidsequence 26 H-CDR3 amino acid sequence 27 Light chain amino acidsequence 28 VL nucleotide sequence 29 VL amino acid sequence 30 L-CDR1amino acid sequence 31 L-CDR2 amino acid sequence 32 L-CDR3 amino acidsequence 33 General BBB transporter VH amino acid sequence 34 VL aminoacid sequence 35 L-CDR1 amino acid sequence 36 L-CDR2 amino acidsequence 37 L-CDR3 amino acid sequence 38 BBBt0626gl VH amino acidsequence 39 H-CDR1 amino acid sequence 40 H-CDR2 amino acid sequence 41H-CDR3 amino acid sequence 42 VL amino acid sequence 43 L-CDR1 aminoacid sequence 44 L-CDR2 amino acid sequence 45 L-CDR3 amino acidsequence 46 BBBt0626 VH amino acid sequence 47 H-CDR1 amino acidsequence 40 H-CDR2 amino acid sequence 41 H-CDR3 amino acid sequence 42VL amino acid sequence 43 L-CDR1 amino acid sequence 44 L-CDR2 aminoacid sequence 45 L-CDR3 amino acid sequence 46 BBBt0632gl VH amino acidsequence 48 H-CDR1 amino acid sequence 49 H-CDR2 amino acid sequence 50H-CDR3 amino acid sequence 51 VL amino acid sequence 52 L-CDR1 aminoacid sequence 52 L-CDR2 amino acid sequence 54 L-CDR3 amino acidsequence 55

Figure legends:

FIG. 1: Schematic of HTRF® assay.

FIG. 2: Comparison of amino acid sequences of asyn0087, aslo0452ngl-3and aslo0543 heavy chain variable region (VH) (SEQ ID NOs: 2, 14 and 24,respectively) and light chain variable region (VL) (SEQ ID NOs: 3, 19,and 30, respectively). The underlined amino acids correspond to theCDRs.

FIGS. 3A-3D: Nucleotide and Amino Acid Sequence of aslo0452 ngl-3. FIGS.3A and 3B show the variable heavy chain and variable light chainnucleotide and amino acid sequences, respectively, of aslo0452 ngl-3.FIG. 3A discloses SEQ ID Nos: 13 and 14, respectively, in order ofappearance. FIG. 3B discloses SEQ ID Nos: 18 and 19, respectively, inorder of appearance. FIGS. 3C and 3D show the alignment of thesesequences to the closest human germline sequences. FIG. 3C shows thealignment of aslo0452 ngl-3 variable heavy chain domain amino acidsequence (SEQ ID NO: 14) to germline IGHV3-23 (SEQ ID NO: 58) and JH6sequences (SEQ ID NO: 59). FIG. 3D shows the alignment of aslo0452 ngl-3variable light chain domain amino acid sequence (SEQ ID NO: 19) togermline IGLV5-45 (SEQ ID NO: 60) and JL2 (SEQ ID NO: 61), 3 sequences.The complementarity determining regions (CDRs) are underlined andlabelled. Differences from the germline are highlighted in bold andoutlined. All non-Vernier residues in the light chain framework regionsare human germline amino acids. Vernier residues (*) have not beenchanged to match the germline amino acids.

FIGS. 4A and 4B: Epitope binding of lead isolate clones using a panel ofα-syn truncates. ELISA wells are coated with a range of commerciallyavailable α-syn truncates representing the various defined regions ofthe protein: 1-140: full length α-syn, 1-60: N-terminal region only,61-140: non-amyloid component of plaques (NAC) plus C-terminal region,1-95: N-terminal and NAC regions, 96-140: C-terminal region only, ANAC:NAC region deleted, NCAP: is an alternatively spliced form of α-synmissing amino acids 103-129 (rPeptide). Primary antibodies used fordetection were (FIG. 4A) Asyn087; and (FIG. 4B) Aslo0452 ngl-3 (blackbars), aslo0543 (light grey bars) and NIP228 isotype matched control(dark grey bars). Binding is detected with either an anti-human IgG Eu³⁺secondary antibody (FIG. 4A) or an anti-human IgG-HRP secondary antibody(FIG. 4B).

FIG. 5: Specificity of aslo0452 ngl-3 and aslo0543 for α-syn Relative toSynuclein Family Members using a DELFIA Epitope Competition assay. Usingthe epitope competition HTRF assay, the specificity of the affinityoptimized aslo0452 ngl-3 and aslo0543 clones for α-syn was determined bytitration of unlabeled α-syn, β-syn and γ-syn. From this IC₅₀ valueswere determined.

FIG. 6: Specificity of aslo0452 ngl-3 and aslo0543 for human, cynomolgusmonkey and rat α-syn using a HTRF Epitope Competition assay. Using theepitope competition HTRF assay, the species cross-reactivity profile ofthe affinity optimized clones was determined in a similar assay bytitration of unlabeled α-syn and derivation of IC₅₀ values for eachspecies of α-syn.

FIGS. 7A and 7B: Representative Flow Cytometry Results demonstratingthat affinity optimized clones bind to Native human α-syn in a Humanneuroblastoma cell line.

FIGS. 7A, 7C, 7E and 7G show binding to α-syn negative human breastcancer cell line, BT20. FIGS. 7B, 7D, 7F and 7H show binding to theα-syn positive human neuroblastoma cell line, SHSY5Y. FIGS. 7A-7D: Theprimary human antibodies used in this study were asyn0087 and Hu IgGcontrol. Human antibody binding was detected using a secondaryanti-Human IgG-FITC (Jackson). FIGS. 7E-7H: The primary human antibodiesused in this study were aslo0452 ngl-3, aslo0543, and NIP228 isotypematched IgG1 TM control. Human antibody binding was detected using asecondary anti-Human IgG-FITC (Jackson). The primary mouse antibodiesused were 4D6 (Covance), and an isotype matched negative control (R&DSystems). Mouse antibody binding was detected using a secondaryanti-mouse IgG-FITC (Sigma).

FIG. 8: Specificity of optimized anti-α-syn IgGs for aggregated humanα-syn by DELFIA ELISA. The graph shows that aslo0452 ngl-3 and aslo0543,two high affinity α-syn specific clones, and the lead antibody asyn0087detected captured aggregated forms of α-syn (black bars) but did notdetect captured monomeric α-syn.

FIGS. 9A-9P: Specificity of affinity optimised clones in diseaserelevant tissues by immunohistochemistry. FIGS. 9A-9P show staining withaslo0452 ngl-3, asyn0087 and aslo0543, respectively. FIGS. 9A-9C showaslo0452 ngl-3 staining both Lewy bodies (FIGS. 9A-9B) and Lewy neurites(FIG. 9C) of the substantia nigra in PD brain tissue. FIG. 9D showsaslo0452 ngl-3 shows low level staining of α-syn in cells from thetemporal cortex in a normal brain section. FIGS. E-G show aslo0452 ngl-3staining Lewy bodies, Lewy neurites and Lewy dots of the amygdala in PDbrain tissue. FIG. H shows an isotype matched control antibodydemonstrating no staining in the amygdala in PD brain tissue. FIG. 9I-9Mshow asyn0087 staining of the Locus Coeruleus in PD brain tissue;pathological features identified are Lewy bodies (FIG. 9I-9L), neuronalaggregates (FIG. J), Lewy neurites (FIG. K), and Pale bodies (FIG. 9M).FIGS. 9N-9O show aslo0543 staining Lewy bodies and Lewy neurites in thesubstantia nigra in PD brain tissue. FIG. 9P shows aslo0543 low levelstaining of α-syn in cells from the temporal cortex in a normal brainsection.

FIGS. 10A and 10B: Systemic administration of aslo0452 ngl-3 rapidlylowers free-asyn levels in prefrontal cortex of rats. Mean±SEM absolute(FIG. 10A) or relative (FIG. 10B) free α-synuclein concentration in ISFof aslo0452 ngl-3 (30 mg/kg intravenously; closed symbols) or vehicle(open symbols) treated rats.

FIGS. 11A and 11B: Aslo0452 ngl-3 dose and time-dependently lowersfree-asyn levels in CSF of rats upon systemic administration. Mean±SEMabsolute (FIG. 11A) or relative (FIG. 11B) free α-synucleinconcentration in CSF of aslo0452 ngl-3 (3, 10, 30, 100 mg/kgintravenously; closed symbols) or vehicle (open symbols) treated rats.

FIGS. 12A-12C: The anti-alpha-synuclein antibodies aslo0452 ngl-3 andaslo0452 ngl-3-D265A block ipsilateral-to-contralateral alpha-synucleinspreading. (FIG. 12A): non-tg mice injected with LV-α-syn into the righthippocampus (black arrows) were passively immunised with weekly doses ofanti-alpha-synuclein mouse IgG1 antibodies: aslo0452 ngl-3, aslo0452ngl-3 D265A, 9E4, or with NIP228 isotype control antibody, for 13 weeks,followed by determination of alpha-synuclein spreading byimmunocytochemistry with SYN-1 and automated image analysis. (FIG. 12B):Quantification of alpha-synuclein immunoreactivity data obtained fromimmunocytochemical analysis of ipsilateral hippocampal coronal sectionsrepresented in A. Each column is the mean±SEM value of 10 independentantibody treatments (n=10 mice per antibody treatment group). *P<0.05 vsNIP228; 1-way ANOVA with Dunnett's post-test. (FIG. 12C): Quantificationof alpha-synuclein immunoreactivity data obtained fromimmunocytochemical analysis of contralateral hippocampal coronalsections represented in panel A. Each column is the mean±SEM value of 10independent antibody treatments (n=10 mice per antibody treatmentgroup). *P<0.05 vs NIP228; 1-way ANOVA with Dunnett's post-test.

FIGS. 13A-13C: The anti-alpha-synuclein antibodies aslo0452 ngl-3 andaslo0452 ngl-3-D265A reduce deposition and dissemination oflentivirally-expressed alpha-synuclein along axons. (FIG. 13A): non-tgmice injected with LV-α-syn into the right hippocampus were passivelyimmunised with weekly doses of anti-alpha-synuclein mouse IgG1antibodies: aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or with NIP228isotype control antibody, for 13 weeks, followed by immunocytochemicalanalysis of alpha-synuclein deposits along ipsilateral and contralateraltrans-hippocampal axons (black arrows). (FIG. 13B): Quantification ofipsilateral axonal alpha-synuclein deposits determined byimmunocytochemistry with SYN-1 and automated image analysis. Each columnis the mean±SEM value of 10 independent antibody treatments (n=10 miceper antibody treatment group). *P<0.05 vs NIP228; 1-way ANOVA withDunnett's post-test. (FIG. 13C): Quantification of contralateral axonalalpha-synuclein deposits determined by immunocytochemistry with SYN-1and automated image analysis. Each column is the mean±SEM value of 10independent antibody treatments (n=10 mice per antibody treatmentgroup). *P<0.05 vs NIP228; 1-way ANOVA with Dunnett's post-test.

FIGS. 14A-14C: The anti-alpha-synuclein antibodies aslo0452 ngl-3 andaslo0452 ngl-3-D265A reduce alpha-synuclein deposition in CA1hippocampal neurons and layer 5 neocortical neurons. (FIG. 14A): Non-tgmice injected with LV-α-syn into the right hippocampus were passivelyimmunised with weekly doses of anti-alpha-synuclein mouse IgG1antibodies: aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or with NIP228isotype control antibody, for 13 weeks, followed by immunocytochemicalanalysis of alpha-synuclein deposits in ipsilateral CA1 hippocampalneurons and ipsilateral layer 5 neocortical neurons (black arrows).(FIG. 14B): Quantification of alpha-synuclein deposits in ipsilaterallayer 5 neocortical neurons determined by immunocytochemistry with SYN-1and automated image analysis. Data shown represents the number ofalpha-synuclein positive cells (neurons) per 0.1 sq mm. Each column isthe mean±SEM value of 10 independent antibody treatments (n=10 mice perantibody treatment group). *P<0.05 vs NIP228; 1-way ANOVA with Dunnett'spost-test. (FIG. 14C): Quantification of alpha-synuclein deposits inipsilateral CA1 hippocampal neurons determined by immunocytochemistrywith SYN-1 and automated image analysis. Data shown represents thenumber of alpha-synuclein positive cells (neurons) per 0.1 sq mm. Eachcolumn is the mean±SEM value of 10 independent antibody treatments (n=10mice per antibody treatment group). *P<0.05 vs NIP228; 1-way ANOVA withDunnett's post-test.

FIGS. 15A-15C: aslo0452 ngl-3 and aslo0452 ngl-3-D265A antibodies blockalpha-synuclein spreading in alpha-synuclein transgenic mice. (FIG.15A): α-syn tg mice injected with LV-α-syn into the right hippocampus(black arrows) were passively immunised with weekly doses ofanti-alpha-synuclein mouse IgG1 antibodies: aslo0452 ngl-3, aslo0452ngl-3 D265A, 9E4, or with NIP228 isotype control antibody, for 13 weeks,followed by determination of alpha-synuclein spreading byimmunocytochemistry with SYN-1 and automated image analysis. (FIG. 15B):Quantification of alpha-synuclein immunoreactivity data obtained fromimmunocytochemical analysis of ipsilateral hippocampal coronal sectionsrepresented in panel A. Each column is the mean±SEM value of 10independent antibody treatments (n=10 mice per antibody treatmentgroup). *P<0.05 vs NIP228; 1-way ANOVA with Dunnett's post-test. (FIG.15C): Quantification of alpha-synuclein immunoreactivity data obtainedfrom immunocytochemical analysis of contralateral hippocampal coronalsections represented in panel A. Each column is the mean±SEM value of 10independent antibody treatments (n=10 mice per antibody treatmentgroup). *P<0.05 vs NIP228; 1-way ANOVA with Dunnett's post-test.

FIGS. 16A-16C: aslo0452 ngl-3 and aslo0452 ngl-3-D265A antibodies reducedeposition and dissemination of lentivirally-expressed alpha-synucleinalong axons in transgenic mice. (FIG. 16A): α-syn tg mice injected withLV-α-syn into the right hippocampus were passively immunised with weeklydoses of anti-alpha-synuclein mouse IgG1 antibodies: aslo0452 ngl-3,aslo0452 ngl-3 D265A, 9E4, or with NIP228 isotype control antibody, for13 weeks, followed by immunocytochemical analysis of alpha-synucleindeposits along ipsilateral and contralateral trans-hippocampal axons(black arrows). (FIG. 16B): Quantification of ipsilateral axonalalpha-synuclein deposits determined by immunocytochemistry with SYN-1and automated image analysis. Each column is the mean±SEM value of 10independent antibody treatments (n=10 mice per antibody treatmentgroup). *P<0.05 vs NIP228; 1-way ANOVA with Dunnett's post-test. (FIG.16C): Quantification of contralateral axonal alpha-synuclein depositsdetermined by immunocytochemistry with SYN-1 and automated imageanalysis. Each column is the mean±SEM value of 10 independent antibodytreatments (n=10 mice per antibody treatment group). *P<0.05 vs NIP228;1-way ANOVA with Dunnett's post-test.

FIGS. 17A-17D: aslo0452 ngl-3 and aslo0452 ngl-3-D265A antibodies reducealpha-synuclein deposition in CA1hippocampal neurons and layer 5neocortical neurons in alpha-synuclein transgenic mice. (FIG. 17A):α-syn tg mice injected with LV-α-syn into the right hippocampus werepassively immunised with weekly doses of anti-alpha-synuclein mouse IgG1antibodies: aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or with NIP228isotype control antibody, for 13 weeks, followed by immunocytochemicalanalysis of alpha-synuclein deposits in ipsilateral CA1 hippocampalneurons and ipsilateral layer 5 neocortical neurons (black arrows).(FIG. 17B): Quantification of alpha-synuclein deposits in ipsilaterallayer 5 neocortical neurons determined by immunocytochemistry with SYN-1and automated image analysis. Data shown represents the number ofalpha-synuclein positive cells (neurons) per 0.1 sq mm. Each column isthe mean±SEM value of 10 independent antibody treatments (n=10 mice perantibody treatment group). *P<0.05 vs NIP228; 1-way ANOVA with Dunnett'spost-test. (FIG. 17C): Quantification of alpha-synuclein deposits inipsilateral CA1 hippocampal neurons determined by immunocytochemistrywith SYN-1 and automated image analysis. Data shown represents thenumber of alpha-synuclein positive cells (neurons) per 0.1 sq mm. Eachcolumn is the mean±SEM value of 10 independent antibody treatments (n=10mice per antibody treatment group). *P<0.05 vs NIP228; 1-way ANOVA withDunnett's post-test. (FIG. 17D): Quantification of alpha-synucleindeposits in contralateral CA1 hippocampal neurons determined byimmunocytochemistry with SYN-1 and automated image analysis. Data shownrepresents the number of alpha-synuclein positive cells (neurons) per0.1 sq mm. Each column is the mean±SEM value of 10 independent antibodytreatments (n=10 mice per antibody treatment group). *P<0.05 vs NIP228;1-way ANOVA with Dunnett's post-test.

FIG. 18 Epitope competition of aslo0452-ngl3-hIgG1TM withBBBt0626gl-ScFv-Bs2-also0452-ngl-3-hIgG1TM demonstrates in a HTRF assaythat the incorporation of a BBB moiety does not alter the bindingspecificity of aslo0452-ngl3-hIgG1TM. Dylight650 labelledanti-alpha-synuclein antibody, aslo0452hgl3-hIgG1TM binds tobiotinylated alpha synuclein which in turn is bound to cyrptate labelledstreptavidin. After excitation of the cyrptate an energy transfer (FRET)occurs and when in the presence of the dylight650 labelledaslo0452hgl3-hIgG1TM, the dylight650 is excited and results in afluorescene. If a competitor IgG is present then the binding of thedylight650 labelled aslo0452 is blocked and excitation of the dylight650labelled aslo0452 is prevented, resulting in a decrease of fluorescentsignal. Unlabelled aslo0452 and Bbbt0626-Bs2-also0452 hIgG1TM both areable to similarly compete dylight650 labelled aslo0452-ngl3-hIgG1TM.

FIG. 19 Mouse brain endothelial cell binding ofBBBt0626gl-BS2-aslo0452-ngl-3-hIgG1TM demonstates effective targetengagement of the BBB moiety when coupled to aslo0452-ngl3-hIgG1TM. FMAT(Fluorescence Micro-volume Assay Technology) or Mirror-ball assaytechnologies have both been used to determine specific binding ofantibodies to brain endothelial cells. This assay measures the bindingof human IgGs to mouse brain endothelial cells (b.End3). B.End3 cellsare bound similarly by Bbbt0626 hIgG1TM,Bbbt0626glscFv-Bs2-aslo0452-hIgG1TM and Bbbt0626glscFv-Bs2-NIP228hIgG1TM, but not by the control antibody NIP228 hIgG1TM. This binding isdetected with a mouse anti-Fc mAb (human specific) which in turn isdetected with an Alexafluor647 labelled goat anti-mouse Fc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the surprising and unexpecteddiscovery of aslo0452 ngl-3 and aslo0543 antibodies. This discovery hasled to a new group of antibodies having characteristics shared by theaslo0452 ngl-3 and aslo0543 antibodies, as well as sub-groups ofantibodies having characteristics of aslo0452 ngl-3 and aslo0543,respectively.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention derives from antibody asyn0087 comprising avariable heavy chain region (VH) of amino acid sequence of SEQ ID NO: 2and a variable light chain region (VL) of amino acid sequence SEQ ID NO:3, as disclosed herewith.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the present invention derives from antibody asyn0087, whereinsaid antibody or antigen-binding fragment has a K_(D) of less than 500nM and binds the same epitope as any one of antibodies asyn0087,aslo0452ngl-3 and aslo0543, described herewith.

Like asyn0087, the aslo0452 ngl-3 and aslo0543 antibodies bind theC-terminal region (residues 96-140) of human α-synuclein. Morespecifically aslo0452 ngl-3 and aslo0543 antibodies or antigen-bindingfragment thereof binds the region comprised between about amino acid 102and about amino acid 130 of human α-synuclein (e.g., SEQ ID NO: 1). Insome embodiments, any of the antibodies or antigen-binding fragmentsdisclosed herein bind the region comprised between about amino acid 120and about 130 of human α-synuclein (e.g., SEQ ID NO: 1). In someembodiments, any of the antibodies or antigen-binding fragmentsdisclosed herein bind an epitope that is not the same epitope as theepitope bound by the 9E4 antibody.

The aslo0452 ngl-3 and aslo0543 antibodies are selective forα-synuclein. The antibody or antigen-binding fragment thereof does notbind to other synuclein family members such as β-synuclein orγ-synuclein. More specifically, the antibody or antigen-binding fragmentthereof is specific for human α-synuclein.

The aslo0452 ngl-3 and aslo0543 antibodies bind to human, rat orcynomolgus alpha-synuclein. The ability of aslo0452 ngl-3 and aslo0543to bind to human, cynomolgus monkey and rat alpha-synuclein isindicative of binding to a different epitope on human alpha-synuclein ascompared to antibodies that do not bind to human, cynomolgus monkey andrat alpha-synuclein. The aslo0452 ngl-3 and aslo0543 antibodies are thuscapable of being used for in vivo safety evaluation and investigation incynomolgus monkey and rat models of disease.

The aslo0452 ngl-3 and aslo0543 antibodies bind to human α-synucleinwith high affinity. The aslo0452 ngl-3 and aslo0543 antibodies bind toalpha-synuclein with a K_(D) of less than 500 picomolar (pM), less than400 pM, less than 300 pM, less than 150 pM, less than 120 pM, less than115 pM, less than110 pM or 106 pM or less as measured for example usingOctet analysis (see, e.g., Example 9). The aslo0452 ngl-3 and aslo0543antibodies bind to alpha-synuclein with a K_(D) of less than 300picoMolar (pM), less than 250 pM, less than 200 pM, less than 150 pM,less than 120 pM, less than 110 pM or 108 pM, less than 100 pM, lessthan 80 pM or 74 pM or less as measured for example using KinExAanalysis (for a reference KinExA analysis protocol, see, e.g., Example9).

The aslo0452 ngl-3 Fab fragment binds to human α-synuclein with highaffinity. The aslo0452 ngl-3 Fab fragment binds to alpha-synuclein witha K_(D) of less than 300 picoMolar (pM), less than 200 pM, less than 180pM, 174 pM or less as measured for example using KinExA analysis (see,e.g., Example 9.3).

The aslo0452 ngl-3 and aslo0543 antibodies bind to native endogenoushuman α-synuclein. The aslo0452 ngl-3 and aslo0543 antibodies bind toaggregates of human α-synuclein. In particular, the antibodies thereforebind to an epitope that is not required for aggregation. The aslo0452ngl-3 and aslo0543 antibodies are capable of sequestering both monomericand aggregated forms of alpha-synuclein. The antibody or antigen-bindingfragment thereof of the invention are capable of binding monomeric andaggregated forms of alpha-synuclein.

The aslo0452 ngl-3 and aslo0543 antibodies bind disease relevant,pathological forms of α-synuclein, e.g. Lewy bodies, Lewy neurites, Lewydots in Parkinson's disease brain tissues. Minimal staining is observedin normal (non-diseased) brain.

The aslo0452 ngl-3 antibody reduces α-synuclein levels in the braininterstitial fluid. In particular, the aslo0452 ngl-3 antibody reducesfree unbound α-synuclein levels in the brain interstitial fluid.

The aslo0452 ngl-3 antibody reduces α-synuclein levels in thecerebrospinal fluid. In particular, the aslo0452 ngl-3 antibody reducesfree unbound α-synuclein levels in the cerebrospinal fluid. The aslo0452ngl-3 antibody reduces α-synuclein spreading in vivo. This novelfunction of inhibiting alpha-synuclein spreading is indicative ofbinding to a different epitope on human alpha-synuclein as compared toantibodies that do not inhibit spreading.

In some embodiments, any of the antibodies or antigen-binding fragmentsthereof disclosed herein have any one or more of the functionalproperties of aslo0452 ngl-3, e.g., any of the aslo0452 ngl-3 functionalproperties recited herein. In some embodiments, any of the antibodies orantigen-binding fragments thereof disclosed herein have any one or moreof the functional properties of aslo0543, e.g., any of the aslo0543functional properties recited herein.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention competes with antibody aslo0452 ngl-3 and/oraslo0543 for binding to human α-synuclein. In another embodiment, theantibody or antigen-binding fragment thereof of the present inventionbinds to the same epitope on human α-synuclein as antibody aslo0452ngl-3 and/or aslo0543.

It can readily be determined whether an antibody or antigen-bindingfragment thereof binds to the epitope of the reference antibody orantigen-binding fragment as defined above. Such methods are a matter ofroutine in the art. For example, an antibody can be compared to anotherby biochemical competition assay whereby, two antibodies (one labelledfor detection purposed and one not) are incubated simultaneously with agiven antigen. If a binding signal is achieved for the labelled antibodythen the two antibodies are said to recognize distinct, non-overlappingepitopes on the protein of interest. If no binding signal is obtainedthen, conversely, they would be characterized as having overlappingepitopes on the protein sequence because binding of one antibodysterically hinders binding of the second antibody. Further, the aminoacid location of a given epitope can also be identified using modifiedproteins such as truncates, linear peptide sequences derived from anantigen's primary amino sequence, species orthologues, and byproteolytic digest and mass spec analysis of an antibody bound to agiven protein. These methodologies serve to generate a region ofinteraction between antibody and antigen.

Additional routine experimentation (such as peptide mutation and bindinganalyses) can be carried out to confirm whether any observed lack ofbinding is in fact due to binding the epitope of the invention or ifsome other phenomenon (such as steric hindrance) is responsible. Suchexperiments can be carried out using ELISA, RIA, Biacore, flow cytometryor other known antibody binding assays.

For example, for fine mapping of a specific epitope, mathematical modelsof the epitope:paratope interface can be derived from data generatedthrough solving the structure of the antigen:antibody complex using ahigh resolution imaging method such as co-crystallization with X-raydiffraction. To confirm the relevance of the mathematical model derived,in terms of identifying key contact residues defining the epitope, pointmutagenesis of the antigen must be performed subsequently and ananalysis of the effect on strength of binding between antigen andantibody caused by such mutations established. Using this combination ofmethods an exact map of key contact residues comprising the epitope canbe established.

Antibodies or antigen-binding fragments thereof which bind to theepitope of the antibody or antigen-binding fragment thereof of theinvention may be generated by producing variants of the antibody orantigen-binding fragment thereof of the invention. Such variantantibodies or antigen-binding fragments thereof may have CDRs sharing ahigh level of identity with the CDRs of the antibody or antigen-bindingfragment thereof of the invention. For instance, in some embodiments,any of the CDRs disclosed herein of any of the antibodies orantigen-binding fragments disclosed herein may differ by 1 or 2 aminoacid residues as compared to any one or more of the specific CDRsequences referred to herein (e.g., any one or more of the CDRs havingSEQ ID NOs: 5, 15, 16, 20, 10 and 21). Additionally, such antibodies mayhave one or more variations (e.g. a conservative amino acidsubstitution) in the framework regions.

In one embodiment, the antibodies or antigen-binding fragments thereofof the invention have variations in the CDR amino acid sequences thatmaintain at least 80%, at least 85%, at least 90%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% and up to 99% sequence identity to the CDRs of antibodyaslo0452 ngl-3.

In particular, conservative amino acid substitutions are contemplated.Conservative replacements are those that take place within a family ofamino acids that have related side chains. Genetically encoded aminoacids are generally divided into families: (1) acidic: aspartate,glutamate; (2) basic: lysine, arginine, histidine; (3) non-polar:alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar: glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. These families can befurther categorised: serine and threonine are an aliphatic-hydroxyfamily; asparagine and glutamine are an amide-containing family;alanine, valine, leucine and isoleucine are an aliphatic family; andphenylalanine, tryptophan, and tyrosine are an aromatic family. Thus, ingeneral one could expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid, will not have a major effect on the binding functionor properties of the resulting antibody, especially if the replacementdoes not involve an amino acid within a CDR site.

In one embodiment, the antibody or antigen-binding fragment thereofaccording to the invention comprises at least one CDR selected from:

-   -   (i) H-CDR1 of SEQ ID NO: 5,    -   (ii) H-CDR2 of SEQ ID NO: 6,    -   (iii) H-CDR3 of SEQ ID NO: 7,    -   (iv) L-CDR1 of SEQ ID NO: 9,    -   (v) L-CDR2 of SEQ ID NO: 10,    -   (vi) L-CDR3 of SEQ ID NO: 11.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention has at least one CDR selected from the CDRs ofantibody aslo0452 ngl-3, i.e. at least one CDR selected from any one ofSEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO:10, SEQ ID NO: 21.

In another embodiment, the CDR3 of the heavy chain of the antibody orantigen-binding fragment thereof of the present invention is CDR3 of theheavy chain of antibody aslo0452 ngl-3; and/or the CDR3 of the lightchain of the antibody or antigen-binding fragment thereof of the presentinvention is CDR3 of the light chain of antibody aslo0452 ngl-3. Thus,in one embodiment, the CDR3 of the heavy chain of the antibody orantigen-binding fragment thereof according to the invention is CDR3 ofSEQ ID NO: 16 of the heavy chain of antibody aslo0452 ngl-3; and/or theCDR3 of the light chain of the antibody or antigen-binding fragmentthereof according to the invention is CDR3 of SEQ ID NO: 21 of the lightchain of antibody aslo0452 ngl-3.

In a further embodiment, the CDR3 of the heavy chain of the antibody orantigen-binding fragment thereof of the present invention is CDR3 of theheavy chain of antibody aslo0452 ngl-3.

In one embodiment, the CDR3 of the light chain of the antibody orantigen-binding fragment thereof of the present invention is CDR3 of thelight chain of antibody aslo0452 ngl-3.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe invention has the six CDRs of antibody aslo0452 ngl-3 i.e. threeheavy chain CDRs having amino acid sequences SEQ ID NO: 5, SEQ ID NO: 15and SEQ ID NO: 16; and three light chain CDRs having amino acidsequences SEQ ID NO: 20, SEQ ID NO: 10 and SEQ ID NO: 21.

The present invention provides an antibody or antigen-binding fragmentthereof of the present invention comprising a variable heavy chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the nucleotide sequence defined by SEQ ID NO: 13 and a variable lightchain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the nucleotide sequence defined by SEQ ID NO: 18.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a variable heavy chain having a nucleotidesequence defined by SEQ ID NO: 13 and a variable light chain having anucleotide sequence defined by SEQ ID NO: 18.

The present invention provides an antibody or antigen-binding fragmentthereof of the present invention comprising a variable heavy chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the amino acid sequence defined by SEQ ID NO: 14 and a variable lightchain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the amino acid sequence defined by SEQ ID NO: 19.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises (i) a variable heavy chain having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence definedby SEQ ID NO: 14 and a variable light chain having at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acidsequence defined by SEQ ID NO: 19, and (ii) the six CDRs of antibodyaslo0452 ngl-3.

The present invention provides an antibody or antigen-binding fragmentthereof according to the invention comprising a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 14 and a variable lightchain having the amino acid sequence of SEQ ID NO: 19.

In one embodiment, the antibody or antigen-binding fragment thereofaccording to the invention has the six CDRs of antibody aslo0543.

Thus, in one embodiment, the antibody, or antigen-binding fragmentthereof according to the invention comprises:

-   -   a) three heavy chain CDRs having sequences:        -   (i) H-CDR1 of SEQ ID NO: 25,        -   (ii) H-CDR2 of SEQ ID NO: 26; and        -   (iii) H-CDR3 of SEQ ID NO: 27, and    -   b) three light chain CDRs having sequences:        -   (i) L-CDR1 of SEQ ID NO: 31,        -   (ii) L-CDR2 of SEQ ID NO: 32; and        -   (iii) L-CDR3 of SEQ ID NO: 33.

In a further embodiment, the antibody, or antigen-binding fragmentthereof according to the invention comprises a variable heavy chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the sequence defined by SEQ ID NO: 14 and a variable light chainhaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the sequence defined by SEQ ID NO: 19 and further comprises:

-   -   a) three heavy chain CDRs having sequences:        -   (vii) H-CDR1 of SEQ ID NO: 25,        -   (viii) H-CDR2 of SEQ ID NO: 26; and        -   (ix) H-CDR3 of SEQ ID NO: 27, and    -   b) three light chain CDRs having sequences:        -   (vii) L-CDR1 of SEQ ID NO: 31,        -   (viii) L-CDR2 of SEQ ID NO: 32; and        -   (ix) L-CDR3 of SEQ ID NO: 33.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a variable heavy chain having a nucleotidesequence defined by SEQ ID NO: 23 and a variable light chain having anucleotide sequence defined by SEQ ID NO: 29.

The present invention also provides an antibody or antigen-bindingfragment thereof of the present invention comprising a variable heavychain having the amino acid sequence of SEQ ID NO: 24 and a variablelight chain having the amino acid sequence of SEQ ID NO: 30.

In a further embodiment, the antibody, or antigen-binding fragmentthereof, comprises a heavy chain having an amino acid sequence definedby SEQ ID NO: 22 and a light chain having an amino acid sequence definedby SEQ ID NO: 28.

The framework region and CDRs or an antibody may be precisely defined(see, Kabat et al. Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services (1991),91-3242, 1991; and Chothia et al. J. MoI. Biol. (1987), 196:901-917,both of which are incorporated herein by reference).

Minor variations in the amino acid sequences of antibody orantigen-binding fragment thereof of the invention are contemplated asbeing encompassed by the present invention, providing that thevariations in the amino acid sequence(s) maintain at least 75%, morepreferably at least 80%, at least 90%, at least 95%, and most preferablyat least 99% sequence identity to the antibody or antigen-bindingfragment thereof of the invention as defined anywhere herein. Inparticular, conservative amino acid replacements are contemplated.

The invention also provides a single chain amino acid sequencecomprising the light chain of an antibody or antigen-binding fragmentthereof of the present invention as defined anywhere herein. Theinvention also provides a single chain amino acid sequence comprisingthe heavy chain of an antibody or antigen-binding fragment thereof ofthe present invention as defined anywhere herein.

Optimal alignment of sequences for comparison may be conducted, forexample, by the local homology alignment algorithm of Smith and Waterman(Smith and Waterman, Adv. Appl. Math. 2 (1981), 484; incorporated hereinby reference), by the algorithm of Needleman & Wunsch (Needleman &Wunsch, J. Mol. Biol. (1970), 48: 443; incorporated herein by reference)by the search for similarity method of Pearson & Lipman (Pearson &Lipman, Proc Natl Acad Sci U S A (1988), 85: 2444; incorporated hereinby reference), by computer implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA—Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705), or by visual inspection(see Current Protocols in Molecular Biology, F. M. Ausbel et al, eds,Current Protocols, a joint venture between Greene Publishing Associates,In. And John Wiley & Sons, Inc. (1995 Supplement) Ausbubel; incorporatedherein by reference).

Examples of algorithms suitable for determining percent sequencesimilarity or identity are the BLAST and BLAST 2.0 algorithms (seeAltschul et al. J. Mol. Biol. (1990), 215(3): 403-410; and“http://www.ncbi.nlm.nih.gov/” of the National Center for BiotechnologyInformation; both of which are incorporated herein by reference).

In one embodiment, the antibody or antigen-binding fragment thereof ofthe invention is isolated. In another embodiment, the antibody orantigen-binding fragment thereof of the invention is purified.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe invention is a monoclonal antibody. In another embodiment, theantibody or antigen-binding fragment thereof of the invention is ahumanised antibody. In yet another embodiment, the antibody orantigen-binding fragment thereof of the invention is a human antibody.

In one embodiment, the antibody or antigen-binding fragment thereof ofthe present invention is an IgA, IgD, IgE, IgM or IgG, such as IgG1,IgG2, IgG3, and IgG4, antibody or antigen-binding fragment thereof.

In another embodiment, the antibody or antigen-binding fragment thereofof the invention has reduced binding affinity to IgG Fc receptors. Thusthe antibody or antigen-binding fragment of the invention has a lowimmunogenic effect. In one embodiment, the antibody or antigen-bindingfragment thereof is an IgG1 TM antibody or antigen-binding fragmentthereof. IgG1 TM is a IgG1 triple mutant, which contains 3 pointmutations (L234F/L235E/P331S) in the Fc domain that reduce the bindingaffinity of the antibody or antigen-binding fragment thereof to Fc-gammareceptors (FcγRs) (Oganesyan et al. Acta Crystallogr D Biol Crystallogr,(2008) 64: 700-704; incorporated herein by reference). In someembodiments, the antibody-mediated prevention of alpha-synucleinspreading by the antibodies of the invention thus does not requireFc-associated effector functions as a key mechanism of action.

Antigen-binding fragments include Fab, Fv, scFv, dAb, Fd, Fab', F(ab')₂or an isolated complementarity determining region (CDR) havingsufficient framework to bind. A Fab fragment may be a monovalentfragment consisting of the VL, VH, CL and CH1 domains. A F(ab')₂fragment may be a bivalent fragment comprising two Fab fragments linkedby a disulfide bridge at the hinge region. A Fc fragment may consist ofthe CH2 and CH3 domains. A Fv fragment may consist of the VL and VHdomains of a single arm of an antibody. A dAb fragment (Ward et al.Nature (1989), 341: 544-546; incorporated herein by reference) mayconsist of a VH domain. An isolated complementarity determining region(CDR) having sufficient framework to bind may be an antigen bindingportion of a variable region.

An antigen binding portion of a light chain variable region and anantigen binding portion of a heavy chain variable region, e.g., the twodomains of the Fv fragment, VL and VH, can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al.Science (1988), 242(4877): 423-426; and Huston et al. Proc Natl Acad SciU S A (1988), 85: 5879-5883; both of which are incorporated herein byreference). These are obtained using conventional techniques known tothose with skill in the art, and the portions are screened for utilityin the same manner as are intact antibodies.

Antibodies or antigen-binding fragments thereof of the invention mayhave any or all of the advantageous properties as defined above orcombinations thereof. In particular, antibodies or antigen-bindingfragments thereof of the invention may be selective for alpha-synucleinand be able to slow or prevent cell-to-cell transmission and spreadingof alpha-synuclein in vivo.

The functionality of the resulting antibody or antigen-binding fragmentthereof of the invention and in particular (i) its ability to bind theepitope of alpha-synuclein; and (ii) its ability to slow or preventcell-to-cell transmission and spreading of alpha-synuclein in vivo canreadily be determined by assaying its specific activity using thetechniques described herein in the Examples.

This disclosure provides compositions for delivery of the antibody orantigen-binding fragment thereof according to the invention across theblood-brain barrier (BBB) using a transporter molecule that can crossbrain endothelial cells while associated with said antibody or fragment,e.g., while fused or conjugated to said antibody or fragment. BBBsequences are provided herein.

As used herein, the term “payload” is used as shorthand for antibody orantigen-binding fragment thereof as described herewith whose transportacross the BBB can be facilitated by a transporter molecule as providedherein. In particular embodiments, the “payload” covers the heavy chainvariable region of the antibodies of the invention, more particularlythe heavy chain variable region of also0452 ngl-3 or aslo0543.

A payload can be part of the transporter molecule, e.g., as a fusionpolypeptide, or joined to the polypeptide through disulfide bonds orother covalent bonds. Alternatively the payload can be associated withthe transporter molecule in any way that will allow the transportermolecule to facilitate its transport across the BBB, as furtherdescribed below. In certain aspects the payload remains part of thetransporter molecule following BBB transport, and retains centralnervous system (CNS) activity in that form. Alternatively the payloadcan be associated with the transporter molecule during BBB transport,but in a way that allows it to disassociate with the transportermolecule following BBB transport.

The disclosure further provides methods for the treatment or diagnosisof a disease or disorder of the CNS, in particular an α-synucleinopathy,comprising the use of such transporter molecules associated with anantibody or antigen binding fragment thereof according to the invention.

In certain aspects, this disclosure provides an isolated transportermolecule comprising an immunoglobulin-derived polypeptide. In certainaspects the polypeptide is a mimetic or non-mimetic of the camelidantibody FCS, identified and isolated using Fluorescence Micro-volumeAssay Technology (FMAT) to detect binding to brain microvascularendothelial cells (BMVEC), e.g., mouse B.End3 cells. In certain aspects,the immunoglobulin-derived polypeptide is an antibody or an activefragment thereof, where “active” means that the transporter moleculecan, e.g., bind to BMVEC in one or more species, e.g., mouse BMVEC, ratBMVEC, cynomolgus monkey BMVEC, or human BMVEC, internalize into BMVECof one or more species, and/or cross the blood brain barrier eitheralone, or associated with a payload.

In some embodiments, the BBB transporter molecule is a BBB transportermolecule described in U.S. Provisional Patent Application No.62/094,503, which is incorporated herein by reference in its entirety.In certain aspects, the transporter molecule comprises one or more ofBbbt0241, Bbbt0626, Bbbt0626gl, Bbbt0632, BBBt0632gl Bbbt0654, Bbbt0726,Bbbt0727, Bbbt0732, Bbbt0754, Bbbt0674, Bbbt0755, Bbbt0643, Bbbt0579 orBbbt0671 as described in U.S. Provisional Patent Application No.62/094,503, which is incorporated herein by reference in its entirety.

In a particular embodiment, the BBB transporter molecule is Bbbt0626 orBBBt0632.

In certain embodiments, the BBB transporter molecule is germlined, e.g.,Bbbt0626gl is a germlined version of Bbbt0626, called “Bbbt0626gl”.

In a further particular embodiment, the BBB transporter molecule isBBBt0632gl or Bbbt0626gl.

In certain aspects, the transporter molecule does not bind to BMVEC butis still capable of transporting across the BBB as indicated in in vitrotranscytosis assay.

With reference to the BBB transporter molecules, the VH CDRs sequencesdescribed correspond to the classical Kabat numbering locations, namelyKabat H-CDR1 is at positions 31-35, H-CDR2 is a positions 50-65, andH-CDR3 is at positions 95-102. L-CDR2 and L-CDR3 also correspond toclassical Kabat numbering locations, namely positions 50-56 and 89-97,respectively. As used herein, the terms “L-CDR1” or “light chain CDR1”correspond to sequences located at Kabat positions 23-34 in the VL (incontrast, the classical L-CDR1 location according to the Kabat numberingschema corresponds to positions 24-34).

In certain aspects, the immunoglobulin-derived polypeptide comprisesimmunoglobulin heavy chain complementarity determining regions (CDRs).For example the immunoglobulin-derived polypeptide can include animmunoglobulin heavy chain complementarity-determining region-1(H-CDR1), an immunoglobulin heavy chain complementarity-determiningregion-2 (H-CDR2), an immunoglobulin heavy chaincomplementarity-determining region-3 (H-CDR3). In certain aspects, theimmunoglobulin-derived polypeptide can further comprise, oralternatively comprise, immunoglobulin light chain CDRs. For example,the immunoglobulin-derived polypeptide can include an immunoglobulinlight chain complementarity-determining region-1 (L-CDR1), animmunoglobulin light chain complementarity-determining region-2(L-CDR2), and an immunoglobulin light chain complementarity-determiningregion-3 (L-CDR3).

In certain aspects, the immunoglobulin-derived polypeptide contains anH-CDR1, an H-CDR2, an H-CDR3, an L-CDR1, an L-CDR2, and an L-CDR3 withthe following amino acid sequences, respectively:

-   -   (a) SEQ ID NO: 40 as H-CDR1, SEQ ID NO: 41 as H-CDR2, SEQ ID NO:        42 as H-CDR3, SEQ ID NO: 36 as L-CDR1, SEQ ID NO: 37 as L-CDR2,        and SEQ ID NO: 38 as L-CDR3, wherein the CDRs are similar to        those of Bbbt0626 and Bbbt0626gl;    -   (b) SEQ ID NO: 40 as H-CDR1, SEQ ID NO: 41 as H-CDR2, SEQ ID NO:        42 as H-CDR3, SEQ ID NO: 44 as L-CDR1, SEQ ID NO: 45 as L-CDR2,        and SEQ ID NO: 46 as L-CDR3, wherein the CDRs are identical to        those of Bbbt0626 and Bbbt062gl.

In certain aspects, the immunoglobulin-derived polypeptide contains anH-CDR1, an H-CDR2, an H-CDR3, an L-CDR1, an L-CDR2, and an L-CDR3 withthe following amino acid sequences, respectively:

-   -   (a) SEQ ID NO: 49 as H-CDR1, SEQ ID NO: 50 as H-CDR2, SEQ ID NO:        51 as H-CDR3, SEQ ID NO: 53 as L-CDR1, SEQ ID NO: 54 as L-CDR2,        and SEQ ID NO: 55 as L-CDR3, wherein the CDRs are similar to        those of Bbbt0632gl;    -   (b) SEQ ID NO: 49 as H-CDR1, SEQ ID NO: 50 as H-CDR2, SEQ ID NO:        51 as H-CDR3, SEQ ID NO: 53 as L-CDR1, SEQ ID NO: 54 as L-CDR2,        and SEQ ID NO: 55 as L-CDR3, wherein the CDRs are identical to        those of Bbbt0632gl;

In certain alternative embodiments, one or more CDRs as described aboveare identical to the recited CDRs, except for, e.g., 1, 2, 3, 4, or 5single amino acid deletions, substitutions, or insertions.

In certain embodiments, the transporter molecule as provided above cancross the blood brain barrier.

In certain aspects, the H-CDR1, the H-CDR2, the H-CDR3, the L-CDR1, theL-CDR2, and the L-CDR3 can be situated in immunoglobulin frameworkregions to produce an antibody VH and an antibody VL. In certain aspectsthe framework regions can be human-derived framework regions. In certainaspects the antibody VH and antibody VL are fused together, e.g.,through a flexible peptide linker, to form a scFv molecule. In certainaspects the VH and VL further comprise one or more immunoglobulinconstant domains, e.g., a CH1 domain, a hinge region, a CH3 domain, aCH3 domain, a CL-kappa domain, and/or a CL lambda domain. In certainaspects the one or more immunoglobulin constant domains are derived froma human immunoglobulin, e.g., a human IgG1 immunoglobulin. In certainaspects the VH, VL, and/or constant domains can comprise mutations tofacilitate, e.g., longer or shorter half-life, increased or reducedeffector functions, or the ability to attach a payload molecule eithervia peptide fusion, a disulfide bond, or chemical conjugation.

In certain aspects of the invention are provided antibodies orantigen-binding fragments thereof according to the invention associatedwith a transporter molecule that can cross brain endothelial cells asdescribed herewith.

In particular aspects, this disclosure provides an antibody orantigen-binding fragment thereof according to the invention associatedwith a transporter molecule comprising an immunoglobulin-derivedpolypeptide, where the polypeptide comprises an immunoglobulin heavychain variable region (VH) region and an immunoglobulin light chainvariable region (VL) region. In certain aspects theimmunoglobulin-derived polypeptide comprises sequences provided herein,including:

-   -   (a) a VH amino acid sequence at least 80%, 84%, 85%, 90%, 95%,        96%, 97%, 98%, or 99% identical to SEQ ID NO: 39 and a VL amino        acid sequence at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%,        or 99% identical to SEQ ID NO: 43, where SEQ ID NO: 39 and SEQ        ID NO: 43 encode the VH and VL regions of Bbbt0626gl,    -   (b) a VH amino acid sequence at least 80%, 84%, 85%, 90%, 95%,        96%, 97%, 98%, or 99% identical to SEQ ID NO: 47 and a VL amino        acid sequence at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%,        or 99% identical to SEQ ID NO: 43, where SEQ ID NO: 47 and SEQ        ID NO: 43 encode the VH and VL regions of Bbbt0626,    -   (c) a VH amino acid sequence at least 80%, 84%, 85%, 90%, 95%,        96%, 97%, 98%, or 99% identical to SEQ ID NO: 48 and a VL amino        acid sequence at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%,        or 99% identical to SEQ ID NO: 43, where SEQ ID NO: 48 and SEQ        ID NO: 43 encode the VH and VL regions of Bbbt0632

In certain aspects, this disclosure provides an antibody orantigen-binding fragment thereof according to the invention associatedwith a transporter molecule comprising an immunoglobulin-derivedpolypeptide, where the immunoglobulin-derived polypeptide comprises a VHregion and a VL region, where:

-   -   (a) the VH region comprises SEQ ID NO: 34 and the VL region        comprises SEQ ID NO: 35; or    -   (b) the VH region comprises SEQ ID NO: 39 and the VL region        comprises SEQ ID NO: 43; or    -   (c) the VH region comprises SEQ ID NO: 39 and the VL region        comprises SEQ ID NO: 35; or    -   (d) the VH region comprises SEQ ID NO: 47 and the VL region        comprises SEQ ID NO: 43; or    -   (e) the VH region comprises SEQ ID NO: 47 and the VL region        comprises SEQ ID NO: 35; or    -   (f) the VH region comprises SEQ ID NO: 48 and the VL region        comprises SEQ ID NO: 52

In a further embodiment, the VH and VL regions of the transportermolecule as described above are covalently linked to form a single chainfragment (ScFv).

In certain aspects, the transporter molecule provided herewith hastransporter activity, e.g., it can bind to BMVEC from one or morespecies, e.g., mouse, rat, cynomolgus monkey, or human BMVEC, it caninternalize into BMVEC of one or more species, or it can cross the bloodbrain barrier.

In certain aspects, a transporter molecule as provided herein comprisesan immunoglobulin-derived polypeptide, where the immunoglobulin-derivedpolypeptide comprises an antibody or a BBB-penetrable fragment thereof.

A “BBB-penetrable fragment” as described herein is a fragment of thetransporter molecule that can specifically bind to BMVEC of one or morespecies and cross through BMVEC in vitro or in vivo from the peripheralvasculature into the CNS vasculature. Whether a given fragment is aBBB-penetrable fragment can be tested by a variety of in vitro or invivo assays known to persons of ordinary skill in the art. For example,the transporter molecule can be tested in an in vitro transcytosisassay, in an in vivo assay such as a diuresis assay, as described inU.S. 62/094,503. Other assays that could be used to measure in vivodelivery of payloads across the

BBB include, without limitation, chronic constriction injury (CCI);spared nerve injury model (SNI) or spinal nerve ligation (SNL), all ofwhich can be measured via paw flick, or the Hargreaves method(Hargreaves K, et al., Pain; 1988; 32; 77-88). in certain aspects, atransporter molecule as provided herein can bind to BMVEC from one ormore species, e.g., human, cynomolgus monkey, murine, rat, or bovineBMVEC. Binding can be demonstrated in various ways known to persons ofordinary skill in the art, e.g., in a FMAT assay as described in U.S.62/094,503. In certain aspects, the BMVEC are brain capillaryendothelial cells (BCEC). In certain aspects, a transporter molecule asprovided herein can pass through a monolayer of BCEC in an in vitrotranscytosis assay. In certain aspects, transporter molecule activitycan be demonstrated by visualization of the transporter molecule in theCNS. For example, a tritium-labeled transporter molecule can bedelivered to a subject, e.g., a mouse peripherally, e.g., intravenously,and then visualized in the CNS via quantitative whole body radiography.In certain aspects, the transporter molecule localizes in specificregions of the CNS, e.g., the cortex of cerebellum, the gray matter ofthe cerebrum, the gray matter of the spinal cord, the pons, or acombination thereof.

In certain aspects, a transporter molecule as described herein comprisesan antibody or BBB-penetrable fragment thereof that comprises orconsists of two or more subunits, e.g., a heavy chain or fragmentthereof and a light chain or fragment thereof, where the heavy chain andlight chain are associated, e.g., as a single fusion protein (e.g., ascFv), or as two subunits held together by one or more disulfide bonds.In certain aspects the heavy chain comprises a VH domain or region andthe light chain comprises a VL domain or region.

In one embodiment, the invention provides an antibody or antigen-bindingfragment thereof according to the invention associated to a blood-brainbarrier transporter molecule as described herewith.

In a particular embodiment, the antibody or antigen-binding fragmentthereof according to the invention is associated to a blood-brainbarrier transporter molecule, wherein the transporter molecule is asingle chain fragment (scFv) comprising:

-   -   i. the heavy chain variable region (VH) of BBBt0626gl of SEQ ID        NO: 39 and the light chain variable region (VL) of BBBt0626gl of        SEQ ID NO: 43, or    -   ii. the heavy chain variable region (VH) of BBBt0626 of SEQ ID        NO: 47 and the light chain variable region (VL) of BBBt0626 of        SEQ ID NO: 43,    -   iii. the heavy chain variable region (VH) of BBBt0632gl of SEQ        ID NO: 48 and the light chain variable region (VL) of BBBt0632g1        of SEQ ID NO: 52

In certain aspects the heavy chain further comprises a heavy chainconstant domain, e.g., a CH1 domain, a hinge, a CH2 domain, and/or a CH3domain, or fragment thereof. In certain aspects the heavy chain constantdomain is an IgG constant domain or fragment thereof, e.g., a human IgGconstant domain, e.g., a human IgG1, IgG2, IgG3 or IgG4 constant domain.In certain aspects, the IgG constant domain or fragment thereofcomprises an altered glycosylation and/or one or more amino acidsubstitutions relative to a wild-type IgG constant domain wherein themodified IgG has a particular property, e.g., an increased or decreasedhalf-life compared to the half-life of an IgG having the wild-type IgGconstant domain, either increased or decreased effector functionsrelative to a wild-type IgG constant domain, or the ability to attachheterologous moieties via, e.g., a peptide bond, a disulfide bond, or achemical conjugation. In certain aspects, the IgG constant domain orfragment thereof has an altered glycosylation relative to a wild-typeIgG constant domain wherein the modified IgG has a particular property,e.g., an increased or decreased half-life compared to the half-life ofan IgG having the wild-type IgG constant domain, either increased ordecreased effector functions relative to a wild-type IgG constantdomain.

In some embodiments, the antibody or antigen binding fragment thereofaccording to the invention is associated with BBBt0626 or BBBt0626gl, asdefined herewith, forming bispecific antibody molecules.

In other embodiments, said bispecific antibodies according to theinvention comprise the human IgG1 TM backbone (i.e. the heavy chain CH1,CH2, CH3 regions of an IgG1 TM) associated with a single chain fragment(scFv) comprising the VH and VL regions of BBBt0626 or BBBt0626glgrafted to the N-terminus (“BiS2 format”) or C-terminus (“BiS3 format”)of the heavy chain or N-terminus (“BiS1 format”) of the VL, of ananti-α-synuclein antibody according to the invention. BiS formatreferences are as disclosed in DiMasi et al. J Mol Biol. 2009 Oct. 30;393(3):672-92.

In some embodiments, said bispecific antibodies according to theinvention further comprise a light chain comprising the kappa or lambdaCL region associated with the VL of an anti-α-synuclein antibodyaccording to the invention.

In particular embodiments, the bispecific antibodies according to theinvention comprise the human IgG1 TM backbone associated with:

-   -   (i) a single chain fragment (scFv) of BBBt0626gl comprising the        heavy chain variable region (VH) of SEQ ID NO: 39 and the light        chain variable region (VL) of SEQ ID NO: 43; or    -   (ii) a single chain fragment (scFv) of Bbbt0626 comprising the        heavy chain variable region (VH) of SEQ ID NO: 47 and the light        chain variable region (VL) of SEQ ID NO: 43,    -   wherein said ScFv is grafted to the N-terminus (BiS2 format) or        C-terminus (BiS3 format) of the heavy chain of aslo0452 ngl-3 of        SEQ ID NO: 12 or N-terminus (“BiS1 format”) of the light chain        of SEQ ID NO. 17.

In still further particular embodiments, the bispecific antibodiesaccording to the invention comprise the human IgG1 TM backboneassociated with a single chain fragment (scFv) of BBBt0626gl comprising(i) the heavy chain variable region (VH) of SEQ ID NO: 39 and (ii) thelight chain variable region (VL) of SEQ ID NO: 43; wherein said ScFv isgrafted to the N-terminus (BiS2 format) or C-terminus (BiS3 format) ofthe heavy chain of aslo0452 ngl-3 of SEQ ID NO: 12 or N-terminus (“BiS1format”) of the light chain of SEQ ID NO. 17.

In other particular embodiments, the bispecific antibodies according tothe invention comprise the human IgG1 TM backbone associated with:

-   -   (i) a single chain fragment (scFv) of BBBt0626gl comprising the        heavy chain variable region (VH) of SEQ ID NO: 39 and the light        chain variable region (VL) of SEQ ID NO: 43; or    -   (ii) a single chain fragment (scFv) of Bbbt0626 comprising the        heavy chain variable region (VH) of SEQ ID NO: 47 and the light        chain variable region (VL) of SEQ ID NO: 43,    -   wherein said ScFv is grafted to the N-terminus (BiS2 format) or        C-terminus (BiS3 format) of the heavy chain of aslo0543 of SEQ        ID NO: 22 or N-terminus (“BiS1 format”) of the light chain of        SEQ ID NO. 28.

The present invention also provides the antibody or antigen-bindingfragment thereof of the invention for use as a medicament.

The present invention also provides an antibody or antigen-bindingfragment thereof of the invention for use in prevention or treatment ofa disease of the central nervous system, in particular anα-synucleinopathy. In one embodiment, the α-synucleinopathy is selectedfrom Parkinson's disease (PD), dementia with Lewy bodies (DLB), andmultiple system atrophy (MSA). In a preferred embodiment, theα-synucleinopathy is Parkinson's disease (PD).

The present invention also provides the use of an antibody or theantigen-binding fragment thereof of the invention for the manufacture ofa medicament for preventing or treating a disease of the central nervoussystem, in particular an α-synucleinopathy. In one embodiment, theα-synucleinopathy is selected from Parkinson's disease (PD), dementiawith Lewy bodies (DLB), and multiple system atrophy (MSA). In apreferred embodiment, the α-synucleinopathy is Parkinson's disease (PD).

The invention also provides a method of treating or preventing diseasein a patient, the method comprising administering to the patient anantibody, or antigen-binding fragment thereof of the invention. In oneembodiment, the α-synucleinopathy is selected from Parkinson's disease(PD), dementia with Lewy bodies (DLB), and multiple system atrophy(MSA). In a preferred embodiment, the α-synucleinopathy is Parkinson'sdisease (PD).

In use, the antibody or antigen-binding fragment thereof of theinvention is able to treat or prevent disease progression by inhibitingthe propagation and spreading of alpha-synuclein in vivo. The antibodyor antigen-binding fragment thereof of the invention thus provides adistinct advantage over other therapeutics. The invention also providesa method of slowing or preventing disease progression in a subject inneed thereof, comprising administering the antibody or antigen-bindingfragment thereof of the invention to the patient.

In one embodiment, said method of treating disease comprisesadministering a therapeutically effective amount of the antibody orantigen-binding fragment thereof of the invention. In anotherembodiment, said method of preventing disease or slowing or preventingdisease progression comprises administering a prophylactically effectiveamount of the antibody or antigen-binding fragment thereof of theinvention.

The dosage ranges for administration of the antibody or antigen-bindingfragment thereof of the present invention are those to produce thedesired therapeutic effect. It will be appreciated that the dosage rangerequired depends on the precise nature of the antibody orantigen-binding fragment thereof or composition, the route ofadministration, the nature of the formulation, the age of the patient,the nature, extent or severity of the patient's condition,contraindications, if any, and the judgement of the attending physician.Variations in these dosage levels can be adjusted using standardempirical routines for optimisation.

Suitable dosages are in the range of 1 to 50 mg per kg of body weight.They may be in the range of 5 to 30 mg/kg, 10 to 25 mg/kg, or 15 to 20mg/kg. The unit dosage may be administered daily or less frequently, forexample, weekly or monthly.

Administration may be effected by repeated administrations of theantibody or antigen-binding fragment thereof of the invention, for aprolonged period of time. Administration can be concurrent orsequential, and can be effected in any order.

The prevention or treatment defined herein may be applied as a soletherapy or may involve, in addition to the antibody or antigen-bindingfragment of the invention, administration of other agents or establishedtherapies normally used in the treatment of α-synucleinopathies (such asL-3 ,4-dihydroxyphenylalanine (L-DOPA), dopamine (receptor) agonists,catechol-O-methyltransferase (COMT) inhibitors, and/or monoamine oxidasetype B (MAO-B) inhibitors). The administration of other agents orestablished therapies may be in combination with, or as an adjunct to,or in conjunction with, the antibody or antigen-binding fragment of theinvention and may be by way of simultaneous, sequential or separatedosing of the individual components of the treatment.

The combination treatment may be carried out in any way as deemednecessary or convenient by the person skilled in the art and for thepurpose of this specification, no limitations with regard to the order,amount, repetition or relative amount of the compounds to be used incombination is contemplated.

A therapeutically effective amount refers to the amount or the antibodyor antigen-binding fragment thereof, which when administered alone or incombination to a patient for treating disease, or at least one of theclinical symptoms of disease, is sufficient to affect such treatment ofthe disease, or symptom. The therapeutically effective amount can varydepending, for example, on the antibody and/or symptoms of the disease,the age, weight, and/or health of the patient to be treated, and thejudgment of the prescribing physician. An appropriate therapeuticallyeffective amount in any given instance may be ascertained by thoseskilled in the art or capable of determination by routineexperimentation. A therapeutically effective amount is also one in whichany toxic or detrimental effects of the antibody or the antibody orantigen-binding fragment thereof are outweighed by the beneficialeffects.

A “prophylactically effective amount” is any amount of the antibody orthe antibody or antigen-binding fragment thereof that, when administeredalone or in combination to a patient, inhibits or delays the onset orreoccurrence of disease, or at least one of the clinical symptoms ofdisease. In some embodiments, the prophylactically effective amountprevents the onset or reoccurrence of the disease entirely. “Inhibiting”the onset means either lessening the likelihood of the onset of disease,or preventing the onset of disease entirely.

The present invention also provides pharmaceutical compositionscomprising an antibody, or antigen-binding fragment thereof of theinvention. Accordingly, the present invention provides a pharmaceuticalcomposition comprising an antibody or antigen-binding fragment thereofof the invention, together with a pharmaceutically acceptable excipient.Suitable pharmaceutically acceptable excipients may facilitateprocessing of the active compounds into preparations suitable forpharmaceutical administration.

Pharmaceutical compositions of the invention may be formulated for, butnot limited to parenteral delivery, for example intramuscular,subcutaneous or intravenous. Compositions suitable for intramuscular,subcutaneous or intravenous injection include sterile aqueous solutions.

The pharmaceutical composition may take the form of an aqueous solutionand may include physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Thepharmaceutical composition may additionally or alternatively containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The pharmaceuticalcomposition may be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Optionally, the pharmaceutical compositionmay contain suitable stabilisers or agents which increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

The present invention provides an isolated nucleic acid moleculeencoding the antibody, or antigen-binding fragment thereof of theinvention. The present invention also provides a vector comprising theisolated nucleic acid molecule of the invention. The present inventionfurther provides a host cell comprising the vector of the invention.

Antibodies or antigen-binding fragments of the invention are not limitedto a particular method of generation or production. Thus, the inventionprovides antibodies which have been manufactured from a hybridoma thatsecretes the antibody, as well as antibodies produced from arecombinantly produced cell that has been transformed or transfectedwith a nucleic acid or nucleic acids encoding the antibody. Suchhybridomas, recombinantly produced cells, and nucleic acids form part ofthe invention.

EXAMPLES Example 1: Antibody Production

Anti-α-syn specific antibodies were isolated from phage displaylibraries using a series of selection cycles on recombinant human α-syn(“hu α-syn”), both passively immobilised onto microtitre wells and freein solution. Naïive human single chain Fv (scFv) phage display librariescloned into a phagemid vector based on the filamentous phage M13 wereused for selections (Lloyd et al., Protein Eng Des Sel. (2009),22(3):159-68; and Vaughan et al., Nat Biotechnol. (1996), 14(3); 309-14;both of which are incorporated herein by reference).

A representative number of individual clones from the selection outputsafter two or three rounds of selection described above were screenedinitially as soluble scFv fragments in periplasmic E. coli extracts(Kipriyanov et al. J Immunol Methods (1997) 200: 69-77; incorporatedherein by reference) in a homogeneous FRET (fluorescence resonanceenergy transfer) HTRF® (Homogeneous Time-Resolved Fluorescence, CisbioInternational) assay for binding to soluble human α-synuclein.

An HTRF® assay (FIG. 1) is a homogeneous assay technology that utilisesfluorescence resonance energy transfer between a donor and acceptorfluorophore that are in close proximity (Mathis G Clin Chem (1995) 41:1391-1397.; incorporated herein by reference). This assay was used tomeasure macromolecular interactions by directly or indirectly couplingone of the molecules of interest to a donor fluorophore, e.g. europium(Eu3+) cryptate, and coupling the other molecule of interest to anacceptor fluorophore XL665, (a stable cross linked allophycocyanin).Excitation of the cryptate molecule (at 337 nm) resulted in fluorescenceemission at 620 nm. The energy from this emission was transferred toXL665 in close proximity to the cryptate, resulting in the emission of aspecific long-lived fluorescence (at 665 nm) from the XL665. Thespecific signals of both the donor (at 620 nm) and the acceptor (at 665nm) were measured, allowing the calculation of a 665/620 nm ratio thatcompensates for the presence of colored compounds in the assay.

Unpurified anti-α-syn scFv samples were tested for binding tobiotinylated α-syn. 5 microlitres of a solution containing 40 nMbiotinylated human α-syn combined with 0.8 nM streptavidin terbium(Cisbio International, 610SATLB) was added to a 384 well low volumeassay plate (Costar, 3676). Next, 10 microlitres of each dilution ofantibody test sample was added to plate. Finally 5 microlitres of asolution containing DC anti-myc (Cisbio International, 661MYCDAB) wasadded to assay plate. All dilutions were performed in assay buffercontaining 0.8 M potassium fluoride (BDH 103444T) and 0.1% bovine serumalbumin (BSA, Sigma A9576) in Dulbeccos PBS (Invitrogen, 14190185).Assay plates were incubated for 3 hour at room temperature followed by16 hour at 4° C. before reading time resolved fluorescence at 620 nm and665 nm emission wavelengths using an EnVision plate reader (PerkinElmer).

Data were analysed by calculating the 665/620 nm ratio followed by the %Delta F values for each sample. The 665/620 nm ratio was used to correctfor sample interference using the equation below:

${665\text{/}620\mspace{14mu} {nm}\mspace{14mu} {ratio}} = {\left( \frac{665\mspace{14mu} {nm}\mspace{14mu} {signal}}{620\mspace{14mu} {nm}\mspace{14mu} {signal}} \right) \times 10\text{,}000}$

The % Delta F for each sample was then calculated using the equationbelow:

${{DeltaF}\mspace{14mu} (\%)} = {\left( \frac{{{sample}\; 665\text{/}620\mspace{14mu} {nm}\mspace{14mu} {ratio}} - {{negativecontrol}\; 665\text{/}620\mspace{14mu} {nm}\mspace{14mu} {ratio}}}{{negativecontrol}\; 665\text{/}620\mspace{14mu} {nm}\mspace{14mu} {ratio}} \right) \times 10\text{,}000}$

The negative control (non-specific binding) was defined by replacingtest sample with non-tagged human or rat α-syn.

The % Delta F values were subsequently used to calculate % specificbinding as described in the equation below:

${\% \mspace{14mu} {specific}\mspace{14mu} {binding}} = {\left( \frac{\left( {{{Sample}\mspace{14mu} {Delta}\mspace{14mu} F\%} - {{NSB}\mspace{14mu} {Delta}\mspace{14mu} F\%}} \right)}{\left( {{{Total}\mspace{14mu} {binding}\mspace{14mu} {DeltaF}\%} - {{NSB}\mspace{14mu} {DeltaF}\%}} \right)} \right) \times 100}$

IC₅₀ values were determined using GraphPad Prism software by curvefitting using a four-parameter logistic equation:

Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((LogEC50−X)*HillSlope))

-   -   X is the logarithm of concentration.    -   Y is specific binding    -   Y starts at Bottom and goes to Top with a sigmoid shape.

Single chain Fv clones which bound to human α-syn as unpurifiedperiplasmic extracts were subjected to DNA sequencing (Osbourn et al,Immunotechnology (1996), 2: 181-196; and Vaughan et al., Nat Biotechnol.(1996), 14(3); 309-14; both of which are incorporated herein byreference). Unique scFv were expressed again in bacteria and purified byaffinity chromatography (as described in WO 01/66754; incorporated byreference). The potencies of these samples were determined by atitration of the purified preparation for binding to biotinylated humanα-syn in the HTRF assay as described above. Purified scFv preparationsthat exhibited the strongest α-syn interaction were selected forconversion to IgG format.

By titrating antibody in the HTRF assay, clones were ranked for strengthof binding to α-syn. The best α-syn binders were analysed further forboth kinetics of binding to α-syn (k_(off)) as IgGs on an Octet Redbiosensor (see method as described in Example 9), as well as forsynuclein family member selectivity (human α-syn, β-syn, γ-syn) (seemethod as described in Example 4) and for cross-reactivity with murineα-syn (see method as described in Example 5).

Example 2: Derivation of aslo0452 ngl-3

A C-terminally reactive α-syn specific clone, asyn0087, was identifiedby screening for binding to human α-syn in a DELFIA assay (see method asdescribed in Example 4). Asyn0087 binds specifically to human,cynomolgus monkey and rodent α-syn (see method as described in Example5). Asyn0087 was reverted to the closest possible human germlinesequence (Tomlinson VBASE. MRC Centre of Protein Engineering, Cambridge,UK. 1997; incorporated herein by reference) that did not affect potencyby standard mutagenesis techniques prior to optimisation. Followinggermlining, the clone was reassessed for binding to α-syn. Nodetrimental effects were observed.

Large scFv-phage libraries derived from the lead clone were created byoligonucleotide-directed mutagenesis of the variable heavy (VH)complementarity determining regions (CDR) 2 and 3 and light (VL) chainCDRs 1 and 3 using standard molecular biology techniques as described inClarkson and Lowman (2004) (Phage display: A practical approach. Oxford:Oxford University Press; incorporated herein by reference). Thelibraries were subjected to affinity-based phage display selectionsperformed on soluble biotinylated human α-syn in order to selectvariants with higher affinity for human α-syn. The selections wereperformed essentially as described previously in above with theexception of lowering the concentration of soluble biotinylated humanα-syn for each round of selection performed.

Representative clones from each selection output were screened initiallyas soluble scFv fragments in periplasmic E. coli extracts in a HTRFassay for their ability to compete for binding to soluble α-syn againstthe parental α-syn binding clone asyn0087. The performance of eachlibrary in these population screens was used to inform which CDRmutagenesis libraries were added together genetically, or ‘recombined’to create new libraries, and these recombined libraries were subjectedto further rounds of affinity driven selections performed on solublebiotinylated human α-syn.

For both individual and recombined mutagenesis library derived clones,after sequence analysis of positive binders, the clones were expressedand purified as both scFv fragments and IgG and binding to soluble α-synreconfirmed by the epitope competition HTRF assays. By titratingantibody in the HTRF epitope competition assay clones were ranked bytheir IC₅₀ values for relative improvement of binding to α-syn comparedto the parental IgG asyn0087. The best α-syn binders were analysedfurther for synuclein family member selectivity (human α-syn, β-syn,γ-syn) and for cross-reactivity with cynomolgus and rat α-syn by botheither direct binding HTRF assay or epitope competition HTRF assay.

From these iterative rounds of library recombination and screening twopotent α-syn specific, cynomolgus and rat α-syn cross-reactive cloneswere identified, aslo0452 ngl-1 and aslo0467.

Single point mutagenesis was performed on aslo0452 ngl-1 for each of theCDRs where positive improvements in IC₅₀ potency had been observed. Eachposition in the chosen CDR was mutated individually through all 20possible amino acid residues, and again screened by epitope competitionHTRF assay for improved IC₅₀ compared to aslo0452 ngl-1 IgG. Multipleresidues across four CDRs (H2, H3, L1 and L3) were identified andcombined on both aslo0452 ngl-1 and aslo0467, and again assessed byepitope competition HTRF assay for improved IC₅₀ compared to aslo0452ngl-1. From these experiments the two most improved binders wereidentified as aslo0452 ngl-3 and aslo0543.

FIG. 2 compares the amino acid sequences of the VH and VL regions ofasyn0087, aslo0452 ngl-3 and aslo0543.

Example 2.1: Reformatting of scFv to IgG1 TM

Single chain Fv clones with desirable α-syn binding properties wereconverted to effector function null whole immunoglobulin G1 TM (IgG1 TM)(Oganesyan et al. Acta Crystallogr D Biol Crystallogr. (2008), 64(Pt6):700-4; incorporated herein by reference) antibody format essentiallyas described by Persic et al. (Persic et al, Gene (1997) 187: 9-18;incorporated herein by reference) with the following modifications. AnOriP fragment was included in the expression vectors to facilitate usewith CHO-transient cells and to allow episomal replication. The variableheavy (VH) domain was cloned into a vector (pEU1.4) containing the humanheavy chain constant domains and regulatory elements to express wholeIgG1 TM heavy chain in mammalian cells. Similarly, the variable light(VL) domain was cloned into a vector (pEU4.4) for the expression of thehuman light chain (lambda) constant domains and regulatory elements toexpress whole IgG light chain in mammalian cells. To obtain IgGs, theheavy and light chain IgG expressing vectors were transfected intoCHO-transient mammalian cells (Daramola et al, Biotechnol Prog (2014)30: 132-141; incorporated herein by reference). IgGs were expressed andsecreted into the medium. Harvests were filtered prior to purification,then IgG was purified using Protein A chromatography. Culturesupernatants were loaded on a column of appropriate size of CeramicProtein A (BioSepra) and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl.Bound IgG was eluted from the column using 0.1 M Sodium Citrate (pH 3.0)and neutralized by the addition of Tris-HCl (pH 9.0). The elutedmaterial was buffer exchanged into PBS using Nap10 columns (Amersham,#17-0854-02) and the concentration of IgG was determinedspectrophotometrically using an extinction coefficient based on theamino acid sequence of the IgG (Mach et al, Anal Biochem (1992) 200:74-80; incorporated herein by reference). The purified IgG were analyzedfor aggregation and degradation purity using SEC-HPLC and by SDS-PAGE.

The rationale for using IgG1 TM as the candidate drug format is tominimise bystander killing due to immune cell and complement activation(i.e., excessive production of C3a which may cause inflammation).Bystander cell killing may be triggered by the potential accumulation ofimmune complexes formed by the candidate drug and extracellularα-synuclein which has been demonstrated to interact with lipid membranes(Bartels et al., Biophys. J. (2010), 99: 2116-2124; incorporated hereinby reference). The IgG1 TM format was chosen as this has beendemonstrated to have negligible binding to Fcγ receptors (FcγR) andreduced C1q-mediated complement activation by immune complexes(Oganesyan et al. Acta Crystallogr D Biol Crystallogr. (2008), 64(Pt6):700-4; incorporated herein by reference).

To minimise any potential risk of immunogenicity, the frameworks ofaslo0452 ngl-3 are as close to the human germline amino acid sequence asis possible without affecting potency. This means that some amino acidsin aslo0452 ngl-3, including the Vernier residues (Foote and Winter, JMol Biol. (1992), 224(2): 487-99; incorporated herein by reference), arenot mutated to the closest human germline sequence. For the V_(H) domainof aslo0452 ngl-3 there is one Vernier residue in the V region that isnot mutated to the human germline IGVH3-23 and IGJH6 sequence (FIG. 3C).For the V_(L) domain of aslo0452 ngl-3 all framework residues match thehuman germline IGLV5-45 and IGJL2 or IGJL3 sequence (FIG. 3D).

Example 3: Affinity Optimized Anti-α-syn IgG Epitope Confirmation

Recombinant human alpha-, beta-, and gamma-synuclein, recombinanttruncated versions of human alpha-synuclein (aa1-60, aa1-95, aa61-140,96-140, ANAC, and NCAP), and mouse alpha-synuclein were obtained fromrPeptide LLC. Crude epitope mapping was performed using the commerciallyavailable α-syn truncates.

Briefly, one microgram per milliliter of each truncate was coated to amicrotitre well overnight at 4° C. After rinsing the wells in PBS, a 1μg/m1 dilution of each anti-α-syn antibody was added. Following 1 hincubation and washing the bound antibody was detected by addition of ananti-human IgG conjugated to either HRP or Eu³⁺. Subsequent toincubation and washing, the appropriate detection substrate was added(TMB or DELFIA Enhancer solution respectively) and the plate read on amicrotitre plate reader.

These epitope binding studies revealed that the lead isolate, asyn0087,recognizes an epitope located in the C-terminal region of the α-synprotein between amino acids 102 and 130 (FIG. 4A). Both aslo0452 ngl-3and aslo0543 maintain their recognition of the same epitope located inthe C-terminal region of the α-syn protein between amino acids 102 and130 as their parental lead isolate asyn0087 (FIG. 4B).

Example 4: Specificity of aslo0452 ngl-3 and aslo0543 for α-syn Relativeto Synuclein Family Members Using an Epitope Competition HTRF Assay

It is important for an antibody intended to be used in therapeuticapplications to be specific for human α-syn, in order to minimize anypotential safety risks related to off target interaction with the othersynucleins (β-synuclein and γ-synuclein).

The specificity of aslo0452 ngl-3 and aslo0543 for α-syn over the othersynuclein family members, β-syn and γ-syn, was determined using the HTRFepitope competition assay that measured the binding of biotinylatedhuman α-syn to the antibody in solution.

α-syn, β-syn and γ-syn were titrated into the assay, and the selectivityof aslo0452 ngl-3 IgG and aslo0543 IgG was assessed by measuring thedegree of inhibition of biotinylated human α-syn binding to aslo0452ngl-3/aslo0543. IC₅₀ values were determined by curve fitting the data toa four parameter logistic equation using PRISM 6® software (Graphpad).More sensitive HTRF assays measuring direct binding of IgG to humanα-syn, β-syn and γ-syn were also used to confirm α-syn specificity (datanot shown). For the negative control, the antibody test sample wasreplaced with an isotype control antibody or buffer only.

Representative IC₅₀ values obtained with α-syn, β-syn and γ-syn proteinsin the aslo0452 ngl-3 and aslo0543 HTRF epitope competition assays areshown in FIG. 5. No binding to β-syn and γ-syn was observed at theconcentrations tested (up to 5 μM), demonstrating that aslo0452 ngl-3and aslo0543 are selective for α-syn.

Example 5: Specificity of aslo0452 ngl-3 for Human, Cynomolgus Monkeyand Rat α-syn Using a HTRF Epitope Competition Assay

In view of therapeutic applications, it is important that the antibodyis cross reactive to cynomolgus α-synuclein and desired that it is crossreactive to rat α-synuclein, to within 10-fold of that observed againsthuman α-syn. This is to enable safety studies to be carried out in bothcynomolgus monkey and rat species.

The specificity of aslo0452 ngl-3 and aslo0543 for human, cynomolgusmonkey and rat α-syn was determined using the HTRF epitope competitionassay that measured the binding of biotinylated human α-syn to aslo0452ngl-3 in solution.

Human, cynomolgus monkey and rat α-syn were titrated into the assay, andthe selectivity of the antibody was assessed by measuring the degree ofinhibition of biotinylated human α-syn binding to the antibody. IC₅₀values were determined by curve fitting the data to a four parameterlogistic equation using PRISM 6® software (Graphpad). Species crossreactivity of aslo0452 ngl-3 and aslo0543 was also confirmed using adirect binding HTRF assay format (not shown).

Aslo0452 ngl-3 (or aslo0543) was titrated into the assay to compete forhuman or cynomolgus monkey or rat α-syn binding to aslo0452 ngl-3 (oraslo0543) by HTRF assay. For the negative controls, the antibody testsample was replaced with an isotype control antibody or buffer only.

Representative IC₅₀ values obtained with human, cynomolgus monkey andrat α-syn proteins in the HTRF epitope competition assay are shown inFIG. 6. Aslo0452 ngl-3 binds to human and cynomolgus α-syn with IC₅₀values of 5.7 nM and 6.8 nM respectively, and binds to rat α-syn withIC₅₀ value of 19.6 nM, within 4-fold. Aslo0543 binds to human andcynomolgus α-syn with IC₅₀ values of 2.0 nM and 2.1 nM respectively, andbinds to rat α-syn with IC₅₀ value of 3.8 nM, within 2-fold.

The ability of aslo0452 ngl-3 to bind to human, cynomolgus monkey andrat α-syn is indicative of binding to a different epitope on humanalpha-synuclein as compared to antibodies that do not bind to human, andcynomolgus monkey and rat α-syn.

Example 6: Specificity of Affinity Optimized Clones for Native α-synMeasured by Flow Cytometry

The specificity of the affinity optimized anti-α-syn IgGs, aslo0452ngl-3 and aslo0543, for binding to native, endogenous human α-syn wasdetermined by flow cytometry using α-syn positive and negative celllines.

Briefly, SHSY5Y neuroblastoma cells (α-syn positive) and BT-20 breastcancer cells (α-syn negative) were fixed in 0.01% formaldehyde and thenpermeabilised with 0.5% (v/v) Tween 20 prior to incubation withanti-α-syn antibodies, positive control or isotype control antibodies.After extensive washes, bound antibody was detected by incubation withan anti-human or anti-mouse IgG-FITC secondary antibody. Followingfurther washes the cells were analyzed with a FACS Canto II apparatus(Becton Dickinson, Franklin Lakes, N.J.) and data analysis was performedusing FlowJo Software (Tree Star, Ashland, Oreg.).

Data are plotted as histograms showing the difference between cellsstained alone versus primary antibody stained cells. The results in FIG.7A show a shift in fluorescent signal in the presence of asyn0087 (panelD) in α-syn positive SH-SY5Y cells, compared to isotype control andsecondary antibody alone (panel B), indicating recognition ofendogenously expressed α-syn. Asyn0087 does not bind to the α-synnegative human breast cancer cell line, BT-20 (panel C). The results inFIG. 7B show a strong shift in fluorescent signal in the presence ofeither aslo0452 ngl-3 or aslo0543 (panel H) comparable to the positivecontrol antibody, 4D6 on α-syn positive SH-SY5Y cells (panel F) and noshift on α-syn negative BT-20 cells (panel G). This demonstrates thatboth aslo0452 ngl-3 and aslo0543 bind to native, endogenously expressedintracellular human α-syn.

Example 7: Specificity of Optimized anti-α-syn IgGs for Aggregated Humanα-syn by DELFIA ELISA

Fibrillar preparations or aggregates of human α-syn were generated asdescribed by Emadi et al. (Emadi et al, Biochemistry (2004), 43:2871-2878.; incorporated herein by reference). Briefly, 200 μl of 50 μMrecombinant α-syn was aliquotted into a 1.8 ml Sarstedt tube and placedin a 37° C. shaking incubator for 3 days at 280 rpm. The presence ofaggregated α-syn was determined by incorporation of Thioflavin T addedto a final concentration of 10 μM, incubated in the dark for 1 h at roomtemperature and fluorescence read at an excitation wavelength of 450 nmand emission wavelength of 485 nm on an Envision microplate reader.

The specificity of the affinity optimized anti-α-syn IgGs, aslo0452ngl-3 and aslo0543, and the lead antibody asyn0087, for aggregated humanα-syn was determined using a DELFIA® antibody capture assay. This assaymeasured the capture of aggregated human α-syn by aslo0452 ngl-3,aslo0543 or asyn0087 in a pair-wise ELISA. Briefly, a mouse IgG1 versionof the anti-α-syn antibody was immobilized onto the well of a 96-wellmicrotitre plate (Nunc). After blocking, aggregated or monomeric humanα-syn was incubated in the wells. Following washing, captured humanα-syn was detected by addition of the human IgG1 TM version of the sameanti-α-syn antibody and subsequently an anti-human IgG-Europiumconjugate (Perkin Elmer) or anti-human IgG-HRP conjugate. Subsequent toincubation and washing, the appropriate detection substrate was added(TMB or DELFIA Enhancer solution respectively) and the plate read on amicrotitre plate reader.

In this assay, only aggregated human α-syn will be captured and detectedsince it presents multiple copies of the same epitope on a singleaggregate. For monomeric α-syn only one epitope copy is present,therefore the detecting second antibody cannot bind in the presence ofthe capture antibody. The data are summarized in FIG. 8. The leadisolate, asyn0087, is able to bind to aggregated recombinant humanα-syn. Thus the epitope to which asyn0087 binds is not itself involvedin aggregation of α-syn. Both aslo0452 ngl-3 and aslo0543 retained theirability to bind to aggregated recombinant human α-syn.

Example 8: Specificity of Optimized anti-α-syn IgGs in Disease RelevantTissues by Immunohistochemistry

The specificity of the affinity optimized anti-α-syn IgGs, aslo0452ngl-3 and aslo0543, and the lead antibody asyn0087, for disease relevantforms of human α-syn was determined by immunohistochemical staining ofParkinson's disease brain tissue. The results are shown in FIG. 9 anddemonstrate that like asyn0087, both aslo0452 ngl-3 and aslo0543 canrecognize disease relevant pathological forms of human α-syn inParkinson's disease brain tissue sections, including Lewy bodies, Lewyneurites, neuronal aggregates, Lewy dots and background brain tissue. Nonon-specific staining was observed in normal or healthy brain tissue.

Example 9: Anti-α-syn Antibody Affinity Determination

The equilibrium dissociation constants (KD) for anti-α-syn IgGs forhuman α-syn was determined using two platform technologies: Octet Red(Forte Bio) and KinExA (Sapidyne Instruments).

Both assay systems showed good agreement, indicating that aslo0452 ngl-3affinity was in the sub-nanomolar range. Table 1 shows the affinitymeasurements derived for key anti-α-syn clones generated throughout thelead isolation and lead optimisation process.

Example 9.1: Affinity of aslo0452 ngl-3 by Octet

The affinity of aslo0452 ngl-3 IgG for recombinant bacterially expressedmonomeric human avi-tag α-syn-Flag-His was estimated using an Octet Redinstrument. Aslo0452 ngl-3 was pre-mixed with varying concentrations ofeach ligand until equilibrium was reached. The amount of free antibodywas then measured using the Octet by capturing free aslo0452 ngl-3 usingbiotinylated α-syn immobilized onto streptavidin coated sensors. Theamount of free antibody detected at each α-syn concentration was plottedagainst the concentration of ligand and the KinExA software was used tocalculate the equilibrium dissociation constant (K_(D)). The resultsshown in Table 1 demonstrate that aslo0452 ngl-3 IgG binds to humanα-syn with an affinity of 106 pM.

Example 9.2: Affinity of aslo0452 ngl-3 by KinExA

In addition, the solution phase affinity (K_(D)) of aslo0452 ngl-3 IgGfor recombinant bacterially expressed monomeric human biotinylated α-synwas determined using a KinExA instrument (Sapidyne Instruments).Aslo0452 ngl-3 was pre-mixed with varying concentrations of each liganduntil equilibrium was reached. The amount of free antibody was thenmeasured using the KinExA by capturing free aslo0452 ngl-3 using α-syncoated beads, washing away unbound material and detecting bound antibodyusing a fluorescently labelled species specific antibody. The amount offree antibody detected at each α-syn concentration was plotted againstthe concentration of ligand and the KinExA software was used tocalculate the equilibrium dissociation constant (K_(D)). The resultsshown in Table 1 demonstrate that aslo0452 ngl-3 IgG binds to α-syn withan affinity of 74 pM, showing good agreement with the Octet solutionphase affinity assay above.

Example 9.3: Affinity of aslo0452 ngl-3 Fab Fragment by KinExA

The aslo0452 ngl-3 Fab fragment binds α-synuclein with a high affinity.The K_(D) value of aslo0452 ngl-3 Fab fragment for α-synuclein, asmeasured by KinExA analysis (as described in the above example for thefull antibody), is 174 pM (95% CI: 15-177 pM).

Example 10: Effects of aslo0452 ngl-3 on Free Unbound α-synuclein Levelsin the Prefrontal Cortex Interstitial Fluid (ISF) of Male Sprague DawleyRats

Adult male Sprague Dawley rats (293-417 g; Harlan, the Netherlands) wereanesthetized and guides were implanted in the prefrontal cortex.

One day before the experiment push pull probes (1-3 MDa polyethylenemembrane 4 mm) were implanted in the prefrontal cortex using astereotaxic frame (coordinates for the probes: AP=−3.4 mm (to bregma),lateral+0.8 mm (to midline), ventral−5.0 mm (to dura), the incisor barwas set at −3.3 mm (all coordinates according to Paxinos and Watson. Therat brain in stereotaxic coordinates, Academic Press, New York, 6thedition 2008). The probes were attached to the skull with a stainlesssteel screw and dental cement.

On the day of the experiment the push pull microdialysis probes wereconnected with flexible PEEK tubing (Western Analytical Products Inc.USA; PK005-020) to a microperfusion pump (Harvard) and perfused withartificial CSF (perfusate), containing 147 mM NaCl, 3.0 mM KCl, 1.2 mMCaCl2, and 1.2 mM MgCl2+0.2% BSA at a flow rate of 0.5 μL/min. Theoutlets of the probes were connected to flexible FEP tubing. After aminimum of two hours of pre-stabilization, aslo0452 ngl-3 formulated inPBS or PBS only (vehicle) were dosed at 30 or 0 mg/kg, respectively. Thecompound was administered at 2 mL/kg intravenously. Microdialysissamples were collected at 120 minute intervals. Samples were collectedinto mini-vials (Microbiotech/se AB, Sweden; 4001029). All samples werestored at −80 ° C.

To determine free α-synuclein concentration in rat ISF the microdialysissamples were first subjected to immunoprecipitation to remove aslo0452ngl-3. The immunoprecipitation co-precipitates α-synuclein bound totherapeutic antibodies, while unbound “free” α-synuclein remains in thesupernatant. A solution of protein A beads (Dynabeads® Protein A) wasadded to a 96-well non-skirted plate (polypropylene 0.2 mL) and washedtwice with TBST (50 mM TBS plus 0.1% Tween 20) using a magnet(DynaMag™96 side) to separate the beads from the solution. Thawed ratISF microdialysis samples (10 or 20 μL) were added to each well, mixedwith beads by pipetting up and down and incubated at 4° C. with tiltrotation for 10 minutes. The beads were then pelleted using the magnettwice to ensure complete removal of the beads. The immunoprecipitatedISF samples were transferred to a 96 well plate from an anti-α-synucleinELISA kit (Sensolyte™ Quantitative ELISA kit, human/mouse/rat,AnaSpec,US, AS-55550) with sample diluent buffer already added to atotal volume of 100μL. Calibration samples, 100 μL per well, were addedto the plate in duplicate and 50 μL of detection antibody workingsolution was added to each well. The plate was incubated at +4-8° C.overnight while shaking and protected from light and then washed sixtimes with 350 μL of wash buffer. Finally, 100 μl of TMB coloursubstrate was added to each well and the plate was incubated in 10-15minutes at room temperature in the dark. To stop the reaction, 50 μL ofStop Solution was added to each well and the plate was read within 2hours at an absorbance of 450 nm. Quantification was performed byplotting the response of the standard curve as absorbance units on thelinear scale versus concentration on the logarithmic scale. Afour-parameter function was used for curve fitting.

A time dependent decrease of free α-synuclein was demonstrated in ISF(FIG. 10) after a single intravenous aslo0452 ngl-3 administration of 30mg/kg.

Example 11: Effects of aslo0452 ngl-3 on Free Unbound α-synuclein Levelsin the CSF of Male Sprague Dawley Rats

Adult male Sprague Dawley rats were anesthetized and catheters wereplaced in the cisterna magna to accommodate CSF sampling. A 0.8 cmindwelling cannula was inserted into the cisterna magna, andexteriorized through an incision on top of the skull. The end of the CSFcatheter was fixed in position with dental acrylic cement and attachedto the skull with three stainless steel screws. The animals were alloweda minimum of 2 days of recovery before the compound was administered.

Aslo0452 ngl-3 was formulated in buffer for dosing at 3, 10, 30 or 100mg/kg. The compound or vehicle only was administered at 2 mL/kgintravenously.

After collection of at least four clean CSF samples taken over a minimumof two days, the compound was administered. All animals were dosed witheither aslo0452 ngl-3 or vehicle on day “0”. CSF samples were collectedat each indicated time point. All samples were stored at −80° C. untilshipment.

To determine free α-synuclein in CSF, α-synuclein bound to aslo0452ngl-3, is removed by immunoprecipitation (IP) prior to analysis. The IPwill co-precipitate α-synuclein bound to therapeutic antibodies, whileunbound “free” α-synuclein remains in the supernatant. Determination offree levels of α-synuclein in supernatant is performed using acommercial ELISA kit obtained from Anaspec. The analysis was performedas described for the ISF-results (as described in Example 10).

A dose and time dependent decrease of free α-synuclein was demonstratedin CSF (FIG. 11) after a single intravenous aslo0452 ngl-3administration in the dose range 3-100 mg/kg.

Example 12: Functional Characterisation of aslo0452 ngl-3 by Reductionof Alpha-synuclein Spreading in a Lentiviral In Vivo Model ofAlpha-synucleinopathy

The ability of a high affinity anti-alpha-synuclein antibody, aslo0452ngl-3, to block the spreading of alpha-synuclein was investigated usinga lentiviral in vivo mouse model of alpha-synucleinopathy. For thispurpose, both non-transgenic wild-type mice (non-tg) and alpha-synucleinoverexpressing transgenic mice (α-syn tg) were injected with alentiviral vector that expresses alpha-synuclein (LV-α-syn) into theright hippocampus, and were then passively immunised weekly for 13 weekswith anti-alpha synuclein mouse IgG1 antibodies including aslo0452ngl-3, and an isotype control mouse IgG1 NIP228. At the end of theimmunisation period the mice were euthanised and their brains were fixedin 4% PFA, then coronally sectioned and analysed by immunocytochemistrywith automated image analysis for levels of alpha-synucleinimmunoreactivity ipsilateral and contralateral to the site of theLV-α-syn injection.

Surgery and Passive Immunisation

Three-to-four month old non-transgenic wild type mice (non-tg; n=40) andalpha-synuclein transgenic mice (α-syn tg; n=40) received a singleunilateral injection into the right hippocampus (−2.0, 1.5, −1.3 fromBregma) of a lentiviral vector expressing alpha-synuclein (LV-α-syn).Two weeks after the LV-α-syn injection surgery, mice received weeklydoses of anti-alpha-synuclein mouse IgG1 antibodies: aslo0452 ngl-3(non-tg n=10; α-syn tg n=10), aslo0452 ngl-3 D265A (non-tg n=10; α-syntg n=10), 9E4 (non-tg n=10; α-syn tg n=10), or they were dosed withNIP228 isotype control mouse IgG1 (non-tg n=10; α-syn tg n=10).

All mouse IgGs were dosed at 20 mg/kg by the intraperitoneal (IP) routefor 13 weeks. Animals were group-housed with a maximum or 4 per cage.Animals were kept on 12/12 light/dark cycle with access to food andwater ad libitum. Cages were changed once a week and monitored daily.Any adverse events were reported. All animals tolerated the surgicalprocedures as well as the immunisations. At the end of the antibodytreatment period, mice were euthanised following guidelines for thehumane treatment of animals and their brains were serially sectioned inthe coronal axes and assessed for neuropathological analysis ofalpha-synuclein spreading by immunocytochemical methods.

Alpha-synuclein Immunocytochemistry

Brains were removed, fixed in 4% paraformaldehyde, and sections cut at40 μm intervals in the coronal axes using a vibratome and stored at −30°C. in cryoprotective medium (30% glycerin, 30% ethylene glycol, 40%PBS). Following PBS washes, and blocking buffer steps, sections wereincubated overnight at 4° C. with primary antibody (anti-alpha-synucleinmAb SYN-1 from BD at a dilution of 1:500), washed in PBS and incubatedfor 1 hour at room temperature with secondary antibody (biotinylatedanti-mouse IgG from Vector Laboratories at a dilution of 1:100).Following final PBS wash steps, alpha-synuclein staining was localisedusing the avidin/biotin-peroxidase complex detection system (Elite ABC,Vector Laboratories). Sections were then analysed with automated imageanalysis for levels of alpha-synuclein ipsilateral and contralateral tothe site of the lentiviral vector (LV-α-syn) injection.

Statistics

Data generated from the automated image acquisition of alpha-synucleinlevels across non-tg and α-syn tg treatment groups were analysed usingGraphPad Prism Software, San Diego Calif., USA. One way ANOVA wasperformed with Dunnett's Multiple Comparison post-test. Data shown infigures is presented as mean±standard error of mean (SEM). Differencesbetween groups were considered to be of statistical significance whenp<0.05. All analyses were performed blinded to the rater. The antibodytreatment groups were also blinded to the rater.

Results

In both non-tg mice and α-syn tg mice, alpha-synuclein immunoreactivityon the LV-α-syn-injected ipsilateral side was intense in the neuropiland covered most of the surface of the hippocampus (FIG. 12A, FIG. 15A;NIP228—Ipsilateral). The contralateral non-injected hippocampus ofnon-tg mice and α-syn tg mice also displayed high levels ofalpha-synuclein immunoreactivity, indicating that thelentivirally-expressed alpha-synuclein had spread from the injectedright hippocampus to the left hippocampus (FIG. 12A, FIG. 15A;NIP228—Contralateral). In this lentiviral alpha-synuclein injectionmouse model, previous experiments have shown that only expressedalpha-synuclein protein spreads to the contralateral side with noevidence of transfer of the lentivirus itself, as determined by PCRanalysis (data not shown).

The ipsilateral and contralateral hippocampal levels oflentivirally-expressed alpha-synuclein in non-tg mice that werepassively immunised with 9E4 antibody (9E4—mouse version of PRX002(Prothena)) were almost identical to the ipsilateral and contralateralhippocampal levels of lentivirally-expressed alpha-synuclein in non-tgmice that were passively immunised with NIP228 isotype control mouseIgG1 (FIG. 12 A,B,C; 9E4 compared with NIP228), indicating that 9E4 whendosed at 20 mg/kg weekly for 13 weeks via the IP route does not blockthe dissemination of alpha-synuclein in this alpha-synuclein spreadingmodel.

In contrast, both the ipsilateral and contralateral hippocampal levelsof lentivirally-expressed alpha-synuclein in non-tg mice that werepassively immunised with either aslo0452 ngl-3 antibody or an effectornull mutant version of aslo0452 ngl-3 (aslo0452-ngl-3-D265A), wherereplacement of aspartic acid by alanine at position 265 (D265A) in themouse IgG1 results in loss of interaction between this isotype andlow-affinity IgG Fc receptors (FcγRIIB and FcγRIII) found on microglia,were highly significantly lower than the ipsilateral and contralateralhippocampal levels of lentivirally-expressed alpha-synuclein in non-tgmice that were passively immunised with NIP228 isotype control mouseIgG1 (FIG. 12 A,B,C; aslo0452-ngl-3 & aslo0452-ngl-3-D265A compared withNIP228). This indicates that passive immunisation of mice with eitheraslo0452-ngl-3 or the effector null D265A mutant version ofaslo0452-ngl-3 robustly blocks the spreading of alpha-synuclein in thismouse model of alpha-synucleinopathy. Similar results were obtained whenthe LV-α-syn vector was injected into the right hippocampus of α-syn tgmice; passive immunisation with aslo0452-ngl-3 or aslo0452-ngl-3-D265Abut not 9E4 led to a robust and statistically significant reduction inboth the ipsilateral and contralateral levels of alpha-synucleinimmunoreactivity in the hippocampus when compared to NIP228-treatedα-syn tg mice (FIG. 15 A,B,C).

At higher magnification lentivirally-expressed alpha-synucleinimmunoreactive deposits were observable along both ipsilateral axons ofthe injected side and contralateral axons of the non-injected side ofnon-tg mice (FIG. 13 A; black arrows showing trans-hippocampal axons),suggesting that alpha-synuclein spreading to the contralateralhippocampus may principally occur along axons (trans-axonal spreading).The ipsilateral and contralateral levels of axonal alpha-synucleindeposits in non-tg mice passively immunised with either 9E4 antibody orNIP228 isotype control mouse IgG1 were not significantly different (FIG.13 A,B,C; 9E4 compared with NIP228), indicating that under ourexperimental conditions, 9E4 does not impact the levels of axonalalpha-synuclein deposits nor does it reduce the dissemination ofalpha-synuclein along axons in this lentiviral-alpha-synucleinopathyspreading model.

In contrast, both the ipsilateral and contralateral levels of axonalalpha-synuclein deposits in non-tg mice passively immunised with eitheraslo0452 ngl-3 antibody or the effector null mutant version ofaslo0452-ngl-3 (aslo0452-ngl-3-D265A), were significantly lower than thelevels of axonal alpha-synuclein deposits in non-tg mice treated withNIP228 isotype control mouse IgG1 (FIG. 13 A,B,C; aslo0452-ngl-3 &aslo0452-ngl-3-D265A compared with NIP228), indicating that passiveimmunisation of mice with either aslo0452-ngl-3 or the effector nullD265A mutant version of aslo0452-ngl-3 clears axonal alpha-synucleindeposits and robustly blocks the ipsilateral-to-contralateral transferof alpha-synuclein along axons in this lentiviral-alpha-synucleinopathymouse model.

Very similar results were obtained when the LV-α-syn vector was injectedinto the right hippocampus of α-syn tg mice; passive immunisation withaslo0452-ngl-3 or aslo0452-ngl-3-D265A but not 9E4 led to a robust andstatistically significant reduction in both the ipsilateral andcontralateral levels of alpha-synuclein immunoreactivity along axonswhen compared to NIP228-treated α-syn tg mice (FIG. 16 A,B,C).

In LV-α-syn-injected non-tg mice, at higher magnification intensealpha-synuclein immunoreactivity was detected in the neuropil of theipsilateral hippocampus and to a lesser extent in the neuropil of theipsilateral neocortex (FIG. 14 A). In addition, intense deposits ofalpha-synuclein were detected in a number of identifiable neuronal cellbodies (soma) in the CA1 region of the ipsilateral hippocampus andweaker alpha-synuclein immunoreactivity was detected in layer 5 neuronsof the ipsilateral neocortex (FIG. 14; black arrows). While treatmentwith 9E4 antibody did not significantly alter the number of ipsilateralCA1 hippocampal neurons or ipsilateral layer 5 neocortical neuronscontaining alpha-synuclein deposits when compared to non-tg miceimmunised with NIP228 isotype control mouse IgG (FIG. 14 A,B,C; 9E4compared with NIP228), non-tg mice treated with either aslo0452 ngl-3antibody or an effector null mutant version of aslo0452-ngl-3(aslo0452-ngl-3-D265A) had significantly reduced numbers of ipsilateralCA1 neurons and ipsilateral layer 5 neurons containing alpha-synucleindeposits compared to NIP228 isotype control mouse IgG-treated non-tgmice (FIGS. 14 A,B,C; aslo0452-ngl-3 & aslo0452-ngl-3-D265A comparedwith NIP228).

Also, the intensity of alpha-synuclein immunoreactivity in the neuropiland neurons of the ipsilateral CA1 region of the hippocampus andipsilateral layer 5 region of the neocortex was noticeably lower inaslo0452-ngl-3-treated and aslo0452-ngl-3-D265A-treated non-tg micecompared to NIP228 isotype control mouse IgG-treated non-tg mice (FIG.14A; aslo0452-ngl-3 & aslo0452-ngl-3-D265A compared with NIP228).

Similar results were obtained when the LV-α-syn vector was injected intothe right hippocampus of α-syn tg mice; treatment with aslo0452-ngl-3 oraslo0452-ngl-3-D265A but not 9E4 led to a statistically significantdecrease in the numbers of neurons containing strong alpha-synucleinimmunoreactivity in the ipsilateral CA1 hippocampal and layer 5neocortical regions, as well as in the contralateral CA1 hippocampalregion, when compared to NIP228-treated α-syn tg mice (FIG. 17A, B, C,D).

It has been demonstrated that passive immunisation of either non-tg wildtype mice or α-syn tg mice that have both been stereotactically injectedwith a lentiviral vector that drives expression of human alpha-synucleinon one side of the hippocampus, with a high affinityanti-alpha-synuclein mouse IgG1 antibody aslo0452-ngl-3 robustly reducesthe ipsilateral-to-contralateral trans-axonal spreading of lentivirallyexpressed alpha-synuclein that is observed in this mouse model ofalpha-synuclein propagation. This newly disclosed anti-alpha-synucleinantibody's property of inhibiting alpha-synuclein spreading in vivo isindicative of binding to a different epitope of human alpha-synuclein ascompared to antibodies that do not inhibit spreading in the modeltested, e.g. 9E4 antibody.

Furthermore, the data showing that an effector null D265A mutant versionof aslo0452-ngl-3 is equally effective as aslo0452-ngl-3 at reducingalpha-synuclein spreading in the model indicates that antibody-mediatedprevention of alpha-synuclein spreading does not require Fc-associatedeffector functions as a key mechanism of action, and in particular itindicates that there appears to be no requirement or role for Fcreceptors (FcγRIIB and FcγRIII) present on microglia in theantibody-mediated blockade of alpha-synuclein spreading.

In summary, the antibodies of the invention that target alpha-synuclein,as well as the antigen-binding fragment thereof, have the potential tobe disease-modifying in PD or DLB or MSA by blocking or slowing thepathological uptake of alpha-synuclein into recipient cells andpreventing seeding and transmission of alpha-synuclein pathology betweenanatomically connected brain regions. In this way, antibodies targetingalpha-synuclein may treat or prevent disease progression and be oftherapeutic benefit to patients with synucleinopathies such as PD, DLBor MSA.

Example 13: Generation of 0452 ngl-3-BBBt0626gl Bispecific Antibodies

Exemplary bispecific antibodies according to the invention comprisingthe human IgG1 TM backbone associated with a single chain fragment(scFv) of BBBt0626g1, grafted to the N-terminus (Bis2 format) orC-terminus (Bis3 format) of the heavy chain of aslo0452 ngl-3 weregenerated as described below.

A bispecific antibody according to the invention, in a Bis2 format, wasgenerated by synthetically producing a DNA fragment encodingBbbt0626glscFv-(G4S)x2-aslo0452 ngl-3 VH or Bbbt0626wt-(G4S)x2-aslo452ngl-3 VH that contained BssHII and BstEII flanking endonucleaserestriction sites, upstream of Bbbt0626glscFv or Bbbt0626wt anddownstream of aslo0452 ngl-3 VH, respectively. The digested DNAfragments were then directionally cloned into the hIgG1TM vectorbackbone.

A bispecific antibody according to the invention, in a Bis3 format wasgenerated by PCR amplification followed by directional cloning usingrestriction endonuclease sites (SfiI and XbaI). Two PCR fragments weregenerated: (1) amplifying the hIgG1TM -CH3 domain from the SfiIrestriction site to the C-terminal end of the CH3 domain and (2) an overlapping PCR fragment with a forward PCR oligo incorporating theC-terminal end of the CH3-(G4S)x3 linker (SEQ ID NO: 57) and theN-terminal end of the Bbbt0626gl scFv along with an oligo amplifying theC-terminal end of the Bbbt0626gl scFv and immediate downstream vectorsequence past the XbaI restriction site. Both PCR fragments werestitched together in a pull-through PCR reaction and subsequentlydirectionally cloned into the pEU1_4 (human IgG1TM vector) via the SfiIand XbaI restriction endonuclease sites.

These bispecific antibodies were expressed in a CHO based expressionsystem and the resulting antibodies purified via protein A columnpurification. All Bbbt0626 derived bispecific antibodies were tested forin vitro binding to mouse brain endothelial cell-line (b.end3) toconfirm binding activity of the BBB transporter moiety and also testedfor competition of binding of also0452 ngl-3 in an HTRF based epitopecompetition assay to confirm binding to the aslo0452 ngl-3 epitope.

1-12. (canceled)
 13. An antibody, or antigen-binding fragment thereofthat binds human α-synuclein, wherein the antibody or antigen-bindingfragment thereof comprises: a) three heavy chain CDRs having sequences:(i) H-CDR1 of SEQ ID NO: 25, (ii) H-CDR2 of SEQ ID NO: 26; and (iii)H-CDR3 of SEQ ID NO: 27, and b) three light chain CDRs having sequences:(i) L-CDR1 of SEQ ID NO: 31, (ii) L-CDR2 of SEQ ID NO: 32; and (iii)L-CDR3 of SEQ ID NO:
 33. 14. The antibody, or antigen-binding fragmentthereof, according to claim 13, wherein the antibody or antigen-bindingfragment thereof is an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4antibody or antigen-binding fragment thereof.
 15. The antibody, orantigen-binding fragment thereof, according to claim 13, wherein theantibody or antigen-binding fragment thereof is an IgG1 TM antibody orantigen-binding fragment thereof.
 16. The antibody, or antigen-bindingfragment thereof, according to claim 13, wherein the antibody orantigen-binding fragment thereof. competes with antibody aslo0452 ngl-3for binding to human α-synuclein.
 17. The antibody, or antigen-bindingfragment thereof, according to claim 13, wherein the antibody orantigen-binding fragment thereof. binds to the same epitope on humanα-synuclein as antibody aslo0452 ngl-3.
 18. The antibody orantigen-binding fragment thereof according to claim 13, wherein theantibody or antigen-binding fragment thereof is associated with atransporter molecule for delivery across the blood-brain barrier (BBB).19. The antibody or antigen-binding fragment thereof according to claim18, wherein the transporter molecule is a. an immunoglobulin-derivedpolypeptide comprising BBBt0626gl or a blood-brain barrier-penetrablefragment thereof, or b. BBBt0626 or a blood-brain barrier-penetrablefragment thereof, or c. or BBBt0632gl or a blood-brainbarrier-penetrable fragment thereof.
 20. The antibody or antigen-bindingfragment thereof according to claim 19, wherein the transporter moleculeis a single chain fragment (scFv) comprising: (i) the heavy chainvariable region (VH) of BBBt0626gl of SEQ ID NO: 39 and the light chainvariable region (VL) of BBBt0626gl of SEQ ID NO: 43, or (ii) the heavychain variable region (VH) of BBBt0626 of SEQ ID NO: 47 and the lightchain variable region (VL) of BBBt0626 of SEQ ID NO: 43, or (iii) theheavy chain variable region (VH) of BBBt0632g1 of SEQ ID NO: 48 and thelight chain variable region (VL) of BBBt0632g1 of SEQ ID NO:
 52. 21.(canceled)
 22. The antibody or antigen-binding fragment thereof of claim13, wherein the antibody or antigen-binding fragment thereof binds tohuman α-synuclein with a K_(D) of less than 500 pM.
 23. The antibody orantigen-binding fragment thereof of claim 13, wherein the antibody orantigen-binding fragment thereof reduces α-synuclein spreading in vivo.24. The antibody or antigen-binding fragment thereof of claim 13,wherein the antibody or antigen-binding fragment thereof binds humanα-synuclein but not human β-synuclein or human γ-synuclein.
 25. Theantibody or antigen-binding fragment thereof of claim 13, wherein theantibody or antigen-binding fragment thereof binds to human, rat andcynomolgus α-synuclein.
 26. The antibody or antigen-binding fragmentthereof of claim 13, wherein the antibody or antigen-binding fragmentthereof binds to native endogenous human α-synuclein.
 27. The antibodyor antigen-binding fragment thereof of claim 13, wherein the antibody orantigen-binding fragment thereof binds to aggregates of humanα-synuclein.
 28. The antibody or antigen-binding fragment thereof ofclaim 13, wherein the antibody or antigen-binding fragment thereof bindsdisease relevant, pathological forms of α-synuclein.
 29. The antibody orantigen-binding fragment thereof of claim 13, wherein the antibody orantigen-binding fragment thereof reduces α-synuclein levels, inparticular free unbound α-synuclein, in the brain interstitial fluidand/or in the cerebrospinal fluid.
 30. The antibody or antigen-bindingfragment thereof of claim 13, wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain comprising an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO:
 24. 31. The antibody or antigen-binding fragment thereofof claim 13, wherein the antibody or antigen-binding fragment thereofcomprises a variable heavy chain comprising an amino acid sequence thatis at least 95% identical to the amino acid sequence of SEQ ID NO: 24.32. The antibody or antigen-binding fragment thereof of claim 13,wherein the antibody or antigen-binding fragment thereof comprises avariable heavy chain comprising the amino acid sequence of SEQ ID NO:24.
 33. The antibody or antigen-binding fragment thereof of claim 13,wherein the antibody or antigen-binding fragment thereof comprises avariable light chain comprising an amino acid sequence that is at least90% identical to the amino acid sequence of SEQ ID NO:
 30. 34. Theantibody or antigen-binding fragment thereof of claim 13, wherein theantibody or antigen-binding fragment thereof comprises a variable lightchain comprising an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO:
 30. 35. The antibody orantigen-binding fragment thereof of claim 13, wherein the antibody orantigen-binding fragment thereof comprises a variable light chaincomprising the amino acid sequence of SEQ ID NO:
 30. 36. The antibody orantigen-binding fragment thereof of claim 13, wherein the antibody orantigen-binding fragment thereof is an antibody.
 37. The antibody orantigen-binding fragment thereof of claim 13, wherein the antibody orantigen-binding fragment thereof comprises a L234F/L235E/P331S triplemutation in the Fc region. 38-45. (canceled)
 46. A method of treating orpreventing a disease of the central nervous system (CNS) in a patient,the method comprising administering to the patient an antibody, orantigen-binding fragment thereof, according to claim
 13. 47. The methodaccording to claim 46, wherein the disease is an α-synucleinopathy. 48.The method according to claim 47, wherein the α-synucleinopathy isselected from Parkinson's disease (PD), dementia with Lewy bodies (DLB),and multiple system atrophy (MSA).
 49. The method according to claim 48,wherein the α-synucleinopathy is Parkinson's disease (PD).
 50. Apharmaceutical composition comprising an antibody, or antigen-bindingfragment thereof, according to claim 13, and a pharmaceuticallyacceptable excipient.
 51. An isolated nucleic acid molecule encoding theantibody, or antigen-binding fragment thereof, according to claim 13.52. The isolated nucleic acid molecule of claim 51, wherein the nucleicacid molecule comprises a nucleotide sequence that is at least 90%identical to the nucleotide sequence of SEQ ID NO:
 23. 53. The isolatednucleic acid molecule of claim 51, wherein the nucleic acid moleculecomprises a nucleotide sequence that is at least 90% identical to thenucleotide sequence of SEQ ID NO:
 29. 54. A host cell comprising avector comprising the nucleic acid molecule of claim
 51. 55. Theantibody or antigen-binding fragment thereof of claim 13, wherein theantibody or antigen-binding fragment thereof comprises a variable heavychain comprising an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO: 24, and wherein the antibody orantigen-binding fragment thereof further comprises a variable lightchain comprising an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO:
 30. 56. The antibody orantigen-binding fragment thereof of claim 13, wherein the antibody orantigen-binding fragment thereof comprises a variable heavy chaincomprising the amino acid sequence of SEQ ID NO: 24, and wherein theantibody or antigen-binding fragment thereof further comprises avariable light chain comprising the amino acid sequence of SEQ ID NO:30.
 57. The antibody or antigen-binding fragment thereof of claim 13,wherein the antibody or antigen-binding fragment thereof comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 22 and,wherein the antibody or antigen-binding fragment thereof furthercomprises a light chain comprising the amino acid sequence of SEQ ID NO:28.